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Applied Biosystems ViiA™ 7 Real-Time PCR System User Guide

1. Parameter Description a lt filepath gt Optional Specifies the path and name lt filepath gt of the assay information file aif or aix that the software uses to create new experiment files Example a D assayfiles assayfile aif b lt filepath gt Optional Specifies the path and name lt filepath gt of the bar code file that the software uses to create new files If the b parameter is not used then the software creates the number of experiment specified by the n parameter Example b D barcodefiles barcodefile txt c lt string gt Optional When the parameter is included specifies the alphanumeric string that the software includes in the file names of the new experiments If no value is supplied custom is used as the default value Example c Batch001_ f lt option gt Optional Specifies the convention that the software uses to name the new files The convention can consist of all or some of the following interchangeable arguments in any order e Custom Name Field The alphanumeric string specified by the c parameter e ID The bar code of the plate specified in the bar code file specified by the b parameter Example Custom Name Field_ID If the parameter is used without arguments then the software names files according to the following convention Custom Name Field_ID 1 lt dirpath gt Required Specifies the path of the di
2. Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Sample Name The name of the sample contained by the well 100 character string Detector The name of one target assay applied to the well 100 character string Name if applicable Task Task the target is used for in this well UNKNOWN STANDARD or NTC Quantity The standard quantity if applicable This Float or Integer column only appears for Standard Curve and Relative Standard Curve experiments Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom The ViiA 7 Software can export the data used to generate the multicomponent plot of a real time PCR experiment The multicomponent data tracks the raw fluorescence of all reporter dyes present on the reaction consumable throughout the duration of the experiment run The file begins with a line that names the export format SDS 2 3 and the type of data contained by the file multicomponent A column header occurs next followed by the multicomponent data where each row contains the data for a single well separated by tab characters The multicomponent data contains a dye column for each dye present on the reaction consumable including reporter dyes quencher dyes except non fluorescent dyes and the passive reference Column Description Output Well The number of the well on the consumable Intege
3. 0000 cece eect eee ence eee eee 133 Replace the sample block 2 2 0 0c cece cette eee eens 135 Materials required ass 22 cnee eines ee eden dide abd A E 135 How to handle the sample block 2 0200 cece eee eee eens 135 Replace the sample block 02 cece eee ence eee 135 Replace the heated cover 00 e cece cece eee eee eens 137 Materials required 2 cece cece ccc eee eee eee e ee aee 137 How to handle the heated cover 0 cece cece eee eee eee e eae 137 Replace the heated cover 22 00c cece eee eee ence eee e eee 137 Replace the plate adapter 0c cece cece eee eee eee 139 Materials required 2 020 eee eens 139 Replace the plate adapter 0200 c ccc eee aes 139 APPENDIX A Manual Instrument Operation 00 00eeeeeeeees 141 OVERVIEW orriari tana eee era E EE E deat diane daw Stele dle vey sie bar E O EENE 142 Functions available from the instrument touchscreen 00ee eee eee 142 Operate the instrument from the touchscreen 0 0ece cece eee eee eee 143 Create an experiment from a template 000 cece eee eee 143 Run an experiments s 5scwesG eee Heese a che ee dine eee Aided oes 144 Transfer experiments templates and results data 0 0000000 eee 145 Maintain the instrument from the touchscreen 0 0c cece eee eee 147 Back up and restore the instrume
4. Supplied with the ViiA 7 System Supplies 115 230 V depending on the geographic location of the installation Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 23 Chapter 1 Getting Started ViiA 7 System hardware ViiA 7 System hardware Instrument components Front view Component Description A USB ports Provide USB communication with the ViiA 7 Instrument Can be used to transfer data to and from the instrument and to update the firmware Note If multiple USB drives are plugged into the ViiA 7 Instrument the instrument mounts only the first drive that is installed regardless of the USB port used B Instrument Provides access to the ViiA 7 Instrument functions Can be used to run touchscreen experiments transfer data and operate the instrument functions without the use of the computer C Access door Provides access to the ViiA 7 Instrument lamp the heated cover and the sample block D Lamp Illuminates the reaction plate or array card during a run E Heated cover Covers the plate or array card during a run to prevent condensation and leakage through the consumable cover F Sample block Heats the plate or array card during a run G Side door Opens to allow extension of the tray arm H Plate adapter Secures plates or array cards to the tray arm Tray arm Conveys plates or array cards to and from the sa
5. 0 0000ce cece cece eee e eee eeeeees 37 Archive and back up experiment files 00000 cece cece cece eens 37 Back up the instrument settings 00 cece eect eee eee 37 Fill thevatreay cards titted a R a aad beet e 38 Materials required 00 0 cece cece eeeeeeees 38 Fill the calibration array cards 0 000 cece ccc tee eee ee 38 Perform the ROI calibration 0 00 cece eee nee eee e eee 42 Materials required 0 0 0 cece cece eect ete arr 42 When to perform the calibration 2 00c ccc eee eee ee aee 42 About the ROI calibration data eee een eee eens 43 Prepare the calibration plate or array card 2 2 cece eee 43 Perform the calibration 0 a a e eee eee eee ee eaas 45 Troubleshoot the ROI calibration 0 cece eee eee teen eees 47 Perform the background calibration 0000 cece cette eee ee eeee 48 Materials required 0 0 0 cece rnern rrean nrnna 48 When to perform the calibration 2 0 cece cee eee eens 48 About the background calibration data 0 000 0c eee eee eee 49 Prepare the background plate or array card n n nuanua nnan cece eee eens 49 Perform the calibration ece cece cealee Sec nelle Seeded ede ee eee ee 51 Troubleshoot the background calibration 00000 cece cece eee eee 53 Perform the uniformity calibration 000000 c nnan cece eee eee eee
6. IMPORTANT Do not use the USB ports on the rear panel of the ViiA 7 Instrument The rear USB ports are for use by Applied Biosystems personnel only Transfer templates 1 Plug a USB drive into the USB port on the right side of the touchscreen from a USB drive no 2 If the instrument is in standby touch the ViiA 7 Instrument touchscreen to awaken it then press 3 In the Main Menu touch 72 View Templates 4 In the Browse Experiments screen select the template a Touch then touch USB b Touch the desired template then touch 2 5 In the Save Experiment As screen set the name for the file a Touch the New Template Name field then enter a name for the copied file b Touch the Save to Folder field then select the folder to receive the file c Touch Save 6 Touch to return to the Main Menu 7 Unplug the USB drive Transfer 1 Plug a USB drive into the USB port on the right side of the touchscreen experiments from a E USB drive 2 If the instrument is in standby touch the ViiA 7 Instrument touchscreen to awaken it then press 3 In the Main Menu touch a Browse Experiments 4 In the Browse Experiments screen select the experiment a Touch i then touch USB b Touch the desired experiment then touch 2 5 In the Save Experiment As screen touch the experiment that you want to transfer to the USB drive then touch Save 6 Touch to return to the Main Menu 7 Un
7. IMPORTANT Do not touch the lamp without powder free gloves Finger prints shorten the lamp life 1 Optional Record the lamp installation a Touch the ViiA 7 Instrument touchscreen to awaken it then press b In the Main Menu touch x Tools then touch Record Lamp Installation c Touch the Name field enter any relevant information such as the make model or serial number of the lamp then touch Done d Touch the Comments field then enter any additional information that you want to record such as the make and model of the replacement lamp e When you are finished touch Record New Lamp then touch OK Note The data that you enter for the new lamp appears in the instrument log 2 Power off and unplug the ViiA 7 Instrument then allow it to cool for 15 minutes 3 Open the access door 128 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 5 Service 5 Replace the halogen lamp 4 Remove the lamp from the ViiA 7 Instrument a Slide the lamp release lever downward b Firmly grasp the lamp and lift it up and out of the slotted mount 5 Inspect the lamp for damage carbon usually coats the inside of a failed lamp If necessary dispose of the lamp and obtain a replacement IMPORTANT Dispose of the lamp in accordance with your local municipal waste ordinances Do not dispose of the lamp as unsorted municipal waste
8. Maximum number of wells that can be removed from each standard population d Repeat step 8a through 8c for each replicate population unknowns standards and no template controls on the plate or array card 9 Review the Results Table for quality flags generated by the experiment a Select the Results Table tab b Review the Flag column for wells that generated quality flags c Troubleshoot each well that generated a flag as explained in Troubleshoot the RNase P experiment on page 83 e AMPNC Amplification in negative control e BADROx Bad passive reference signal e BLFAIL Baseline algorithm failed e CTFAIL Cy algorithm failed e EXPFAIL Exponential algorithm failed e HIGHSD High standard deviation in replicate group e NOAMP No amplification e NOISE Noise higher than others in plate e NOSIGNAL No signal in well e OFFSCALE Fluorescence is offscale e OUTLIERRG Outlier in replicate group e SPIKE Noise spikes e THOLDFAIL Thresholding algorithm failed 10 If you omitted outliers click Reanalyze to analyze the run If the status of the RNase P Run is Failed after performing steps 8 through 10 repeat the RNase P experiment using a different RNase P plate If the problem persists contact Applied Biosystems Applied Biosystems ViiA 7 Real Time PCR System User Guide 81 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Verify the instrument performance
9. Spaces in user If you allow spaces in user names and or passwords be aware of the following issues names and or e Leading and trailing spaces in user names are difficult to detect on the screen or in passwords printed reports e The number of consecutive spaces in a user name is difficult to determine on the screen or in printed reports Spaces in user names may cause confusion when a user searches for an audit record associated with a user name To find a record associated with a user name specify the user name exactly including leading consecutive and trailing spaces 104 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators 4 Configure the security system Set up messaging notifications 1 2 In the ViiA 7 Software select Tools gt Security gt Settings In the Security screen click Set Up Messaging Notifications to display the Setup Notifications dialog box Select the events for notification System security enabled or disabled Security has been enabled or disabled User did not enter correct password A user attempts to log in with an incorrect password The message indicates the number of failed authentications User account suspended The user exceeds maximum number of allowed failed authentications login attempts with an incorrect password User session timed out No activity occurred in a user ac
10. ViiA 7 System Command Used to automate the creation of new experiments line Utility and the export of existing experiments Twister II Robot Software Controls the Twister II Robot stores all of the taught positions for the robot and includes the VBA code required to operate the Twister II Robot with the automation control software We recommend that you do not install programs to the C drive Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 1 Getting Started 1 ViiA 7 System software Third party software Before you install third party software to the computer running the ViiA 7 Software confirm that the software will not e Restrict Ethernet communication e Interfere with ViiA 7 Software operation see below To confirm that third party software does not interfere with the ViiA 7 Software 1 Install the software to the computer that contains the ViiA 7 Software 2 Perform several test experiments using dummy plates plates that do not contain reagents Note The goal of the test experiments is to run plates under conditions that match normal instrument operation Therefore the characteristics of the test experiments plate layout and run method must closely resemble your actual experiments 3 Confirm that the ViiA 7 System performs each test experiment without producing errors If the ViiA
11. amplification stage Glossary See assay information file AIF XML version of the assay information file See also assay information file AIF In a diploid organism one of two DNA sequences found at the same locus for example a particular gene but located on homologous chromosomes Two corresponding alleles may have the identical sequence or they may differ somewhat often at one or more single base sites SNPs A segment of DNA amplified during PCR Part of the instrument run in which PCR amplifies the target Fluorescence data collected during amplification are displayed in an amplification plot and the data are used to calculate results Note Only quantitative real time PCR experiments not end point experiments take amplification data into account Calculation of the efficiency of the PCR amplification in an experiment EFF is calculated using the slope of the regression line in the standard curve A slope close to 3 32 indicates optimal 100 PCR amplification efficiency Display of data collected during the cycling stage of PCR amplification The amplification plot can be viewed as e Baseline corrected normalized reporter ARn vs cycle e Normalized reporter Rn vs cycle e Threshold cycle Cr vs well Part of the instrument run in which PCR amplifies the target The amplification stage called a cycling stage in the thermal profile consists of denaturing primer annealing and extension steps
12. e Change the cycle parameter of a stage touch the cycle field modify the setting as desired then touch Close e Delete a stage or step from the thermal profile touch the stage or step you want to remove then touch ge Delete When finished modifying the parameters touch EJ Save In the Save Experiment screen touch each field to set the experiment name reaction volume bar code and any additional information to save to the experiment When finished touch Save amp Start Run to start the experiment In the Start Run screen touch each field as needed to modify the associated parameter then touch Start Run Now to start the experiment Note When the run is complete touch to unload the plate You can download the experiment results to a computer if the ViiA 7 Instrument is connected to a network or you can copy the data to a USB device see Transfer experiments templates and results data on page 145 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix A Manual Instrument Operation A Operate the instrument from the touchscreen Transfer experiments templates and results data You can transfer experiments templates and results data to from the ViiA 7 Instrument using a USB flash drive Before transferring data you must plug the drive into one of the USB ports behind the right side of the ViiA 7 Instrument touchscreen
13. 0 00 ccc ccc cee cence eens 162 Move the VIA 7 Systemes rani ey a ee een een enn eee en neces 163 Materials required onasan un E ei anne deeds aagdes pease deat 163 How to handle the sample block and heated cover 2220000eeeeeeeee 163 Prepare for the ViiA 7 System components 0 cc cece cece ence eens 163 Move the ViiA 7 System 2 0 ccc cece nnn een eee ene e eee eens 164 Reinstall the ViiA 7 System 2 2 ccc nnn ene ence n eens 164 Creating Custom Calibration Plates and Array Cards 165 Create a background plate or array card 2 2 0 cece eee eees 166 Materials required oo 6 heat ceic tad Pied Phe tay lh lege Paroles end 166 Create a background plate 0 cece cece nee tenet eens 166 Create a background array card cee eect annua nanara raaa 167 Create a custom dye plate for calibration 0 00 cece ee eee ee eee eee 168 Before you use custom dyes 00 c cece ect eee e eee 168 Materials required 3 0 2 a a eet word ance tea ce tee a ad Beats Oe 168 Determine optimum dye concentration 20e cece ete eee 168 Create a custom dye plate 0 0 cece eects 170 Add the custom dye to the software 0000 cece eee eee eee eee 171 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Contents APPENDIX D Parts and Materials 53 olcecetoncc daeidene tee thaes estan 1
14. 3 Configure the shortcuts as desired To add a shortcut a Touch the shortcut of interest then touch Set Shortcut b Touch From Templates to link to a specific template file or touch From Folders to link to a folder c Touch the desired template file or folder to configure the shortcut To delete a shortcut touch the shortcut of interest then touch Remove Shortcut or touch Remove All to delete all shortcuts 4 When you are finished configuring the shortcuts touch to return to the Main Menu Review the instrument statistics You can use the ViiA 7 Instrument touchscreen to view usage statistics on the heated cover halogen lamp and other system components View the statistics 1 If the instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press OF 2 In the Main Menu touch ws Tools then touch Show Statistics 3 When you are finished touch fP to return to the Main Menu Applied Biosystems ViiA 7 Real Time PCR System User Guide 155 Calibration Maintenance Networking and Security A Appendix A Manual Instrument Operation Administrate the instrument from the touchscreen Enable or disable instrument security Enable or disable security 156 The ViiA 7 Instrument features a secure mode that can be enabled to restrict local instrument functionality When security is enabled use of the touchscreen is restricted to administrative functions that change the instrument
15. Describes how to store move and reinstall the components of the system Appendix C Creating Custom Calibration Plates and Array Cards Describes how to create a background plate in the event that one is unavailable and how to create a dye plate that can be used to calibrate the system for a dye not manufactured by Applied Biosystems Appendix D Parts and Materials Describes how to order parts accessories and consumables for the ViiA 7 System Appendix E ViiA 7 Software Reference Describes how to use the ViiA 7 Software command line application and provides specifications for files that the ViiA 7 Software imports exports and stores Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security CHAPTER 2 Calibration and Maintenance This chapter covers E Calibrate and maintain the ViiA 7 System 666 34 E About the consumables sic i206 chore bind dade cites EERE cies eds 35 E Perform regular data maintenance 66 6 c cece eens 37 E Fill the array Cards oe cos cise seed soiis sd kura aie i bedai G6 ins Sold gues be unis ileal 38 E Perform the ROI calibration 0 ccc eens 42 m Perform the background calibration 6 c cece eee eee 48 m Perform the uniformity calibration 0 06 c cece eee 55 E Perform the dye calibration 0 c cee eens 60 E Perform the normalization cali
16. Note If you are running a custom dye calibration plate create another plate but decrease the concentration of the dye that exceeded the detectable limit Instrument does not eject the dye plate The adhesive cover may have adhered the plate to the heated cover within the instrument 1 Power off the ViiA 7 Instrument 2 Wait for 15 minutes then power on the ViiA 7 Instrument and eject the plate 3 If the plate does not eject power off and unplug the ViiA 7 Instrument then open the access door 4 Wearing powder free gloves reach into the ViiA 7 Instrument and remove the plate from the heated cover then close the access door 5 Performa background calibration to confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a local Applied Biosystems Field Service Office 68 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the normalization calibration Perform the normalization calibration During the normalization calibration the ViiA 7 System e Collects data from the normalization standards e Stores the information for the normalization standards in a normalization calibration file The normalization calibration generates factors that the ViiA 7 Software uses when comparing data from multiple
17. computer hard drive PlatelD The plate identifier entered into the bar code lt 100 character string gt filed of the experiment Assay Type The type of chemistry application for which the Standard Curve experiment is designed Presence Absence Relative Standard Curve OF DDCt Quantification Run Datetime The date and time that the ViiA 7 Instrument lt date and time gt finished running the experiment Operator The user logged into the ViiA 7 Software atthe lt 100 character string gt time the experiment was run ThermalCycleP The thermal cycling profile for the experiment 96 384 well or arams array card The ViiA 7 Software can export the results data from an analyzed experiment file The format and content of the results data depends on the experiment type and the analysis settings The section begins with a column header followed by the results data where each row contains the data for a single well separated by tab characters If a well contains more than one assay target the ViiA 7 Software lists the data for each additional assay on separate rows repeating the well number and sample information This section describes the following results data formats m Standard Curve Relative Standard Curve and Comparative CT experiments 220 E Genotyping experiments 6 6 ee teeta 221 Applied Biosystems ViiA 7 Real Time PCR System User Guide 219 Calibration Maintenance Networking and Security
18. e Sort the table e Specify filters date range user name action object or record type object or record name reason then click Refresh Note The Reason field in System Configuration History is not used e Select one or more records then click View Report Applied Biosystems ViiA 7 Real Time PCR System User Guide 111 Calibration Maintenance Networking and Security 4 Chapter 4 Security Audit and Electronic Signature Generate audit reports Review the system The System Configuration History lists security audit and electronic signature configuration history configuration records Record type Action Corresponds to Security Settings Update Disable enable or modify security policies session timeout settings Account Settings Update Modify password settings security policies password expiration and account suspension or user name settings User Group Manager Update Create delete or modify reason for change User Role Create Create user role Delete Delete user role Update Modify user role User Account Create Create new user account Update Edit or suspend a user account Role Assignment Delete Assign a different user role to an existing user account Update Create a user account or assign a different user role to an existing account Audit Settings Update Enable or disable auditing Audit Type Update Modify audit settings Function Ma
19. 0 0 7A GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 9 Brain cDNA RGB 0 0 25 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 9 Brain cDNA RGB 0 Eye GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB suoneoyioads pue s ewsoj uodwij 9U 4 J Y JEMYOS 4 VIIA 3 Xpu ddy 4 961 Presence absence experiments q aping J SN W AS S YId WIL IEY 4 V Swalsksolg panddy In 5 pue BuiyJomyay aoueuaquiey uoiesqneg The following example shows a plate setup file created for a presence absence experiment to be run on a ViiA 7 System The experiment screens samples for the presence of a pathogen E coli 0157 H7 The detection assay uses FAM and VIC dye labeled TaqMan probes to amplify a unique genomic sequence and an internal positive control IPC Instrument Type ViiA 7 Passive Reference ROX Sample Setup Well Sample Name Sample Color Biogroup Name Biogroup Color Target Name Target Color Task Reporter Quencher Quantity Comments Control RGB 25 0 0 E coli RGB 98 25 0 NTC FAM NFQ MGB af Control RGB 25 0 0 IPC RGB 98 25 0 NTC VIC NFQ MGB 2 Control RGB 25 0 0 E coli RGB 98 25 0 NTC FAM NFQ MGB 2 Control RGB 25 0 0 IPC RGB 98 25 0 NTC VIC NFQ MGB 3 Control RGB 25 0 0 E coli RGB 98 25 0 NTC FAM NFQ MGB 3 Control RGB 25 0 0 IPC RGB 98 25 0 NTC vic NFQ MGB 4 Pos Control RGB 0 25 0 E coli RGB 98 2570 TPC FAM NFQ MGB 4 Pos Control RGB 0 25 0 IPC RGB 98 25 0 IPC vIc NFQ M
20. 7 Software e Confirm that your computer has an Internet connection e We recommend the following browsers and Adobe Acrobat Reader versions to use the Applied Biosystems website i Microsoft Apple Mozilla Adobe a Operating system Internet Acrobat Safari Firefox Explorer Reader Microsoft Windows v x or later Nonet v2 x or later v4 0 or later Macintosh Nonet v2 0 4 or later t Browser not available for this platform Note Confirm that cookies and Javascript are turned on for the website to function correctly How to order from the ViiA 7 Software 1 To find your assay on the Applied Biosystems Store complete the Find Assay pane in the ViiA 7 Software a Enter a gene name in the Enter Gene Name field then click Find Assay b In the Find Assay Results dialog box select your assay c Click Apply Assay Selection The selected assay gets added to your shopping list 2 Check that the Experiment Shopping List contains the desired materials other than the assay selected in the previous step and that the quantities are correct then click Order Materials in List Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 174 Appendix D Parts and Materials How to order 3 In the Order Materials Login dialog box enter your user name and password for the Applied Biosystems Online Store then click
21. Before removing the plate wait until it reaches room temperature 5 In the ROI dialog box click Finish to complete the calibration then click No when prompted to save the results Applied Biosystems ViiA 7 Real Time PCR System User Guide 169 Calibration Maintenance Networking and Security Appendix C Creating Custom Calibration Plates and Array Cards Create a custom dye plate for calibration Create a custom dye plate IMPORTANT Wear powder free gloves while creating the dye plate 1 Prepare 2 mL of the custom dye at the concentration determined in Determine optimum dye concentration on page 168 2 Pipette 20 uL of the diluted custom dye to all wells of an optical reaction plate 3 Seal the wells of the reaction plate using an optical adhesive cover 4 Centrifuge the plate for 2 minutes at less than 1500 rpm Note The custom dye calibration plate must be well mixed and centrifuged 5 Verify that the liquid in each well of the plate is at the bottom of the well If not centrifuge the plate again at a higher rpm and for a longer period of time Correct Incorrect Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time 170 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix C Creating Custom Calibration Plates and Array Cards C Create a custom dye plate for calibration A
22. Change the Status from SUSPENDED to ACTIVE then click Save Disable inactivate a IMPORTANT You cannot delete a user because user records are required for auditing user account To disable a user account inactivate it as follows 1 Inthe Users screen select the user 2 Click Edit 3 Change the Status from ACTIVE to INACTIVE then click Save Determine the name of the logged in user The title bar of the ViiA 7 Software window displays the name of the user Create or edit a user role User roles determine the permissions associated with a user account The ViiA 7 Software includes three default user roles e Administrator cannot be edited or deleted e Scientist e Technician You can modify the Scientist and Technician roles and you can create your own roles with customized settings as needed To determine the permissions for a default role or to edit it select the role then click Edit Create a user role 1 In the ViiA 7 Software select Tools Security gt Settings 2 In the Security Settings dialog box select the Roles tab 3 Click Create 4 Enter a role name and optional description 5 Select permissions see Permissions and default user roles on page 108 To select all permissions in a category select the check box next to the category Note Operations not shown in the following table are available to all user roles 6 Click Save Role Applied Biosystems ViiA 7 Real Ti
23. If necessary apply pressure as indicated until the adapter snaps into place 5 In the Main Menu touch g 6 Ifyou are installing a tray adapter of a different consumable format replace the sample block if necessary Applied Biosystems ViiA 7 Real Time PCR System User Guide 139 Calibration Maintenance Networking and Security 5 Chapter 5 Service Replace the plate adapter 140 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security APPENDIX A Manual Instrument Operation This appendix covers m Operate the instrument from the touchscreen 00 c ee eee eee eee 143 Create an experiment from a template 00 143 Run an experiments oi ead ees ew eels ena ds waives A e aia aed dared wale bare 144 Transfer experiments templates and results data 0000 005 145 m Maintain the instrument from the touchscreen 0 000 e eee ee eee 147 Back up and restore the instrument settings 00 cece cece 148 Perform an instrument self test 0 ce eee eee 149 Update the instrument firmware 0 eee 150 m Administrate the instrument from the touchscreen 0008 151 Define the date and time 0 cece eee eens 152 Define the instrument settings 066 c ta aot RES ap eee 152 Define the maintenance reminders 0 e cee eee 153 Define the network settings 6 66 cec
24. In the amplification plot a cycle to cycle range that defines background fluorescence This range can be set manually on an assay by assay basis or automatically to set each individual well The magnitude of normalized fluorescence signal generated by the reporter In experiments that contain data from real time PCR the magnitude of normalized fluorescence signal generated by the reporter at each cycle during the PCR amplification In the ARn vs Cycle amplification plot ARn is calculated at each cycle as ARn cycle Rn cycle Rn baseline where Rn normalized reporter See also normalized reporter Rn Expression estimation algorithm Cy which subtracts a baseline component and sets a fluorescent threshold in the exponential region for gene quantification Reactions that contain identical components and volumes but evaluate separate samples of the same biological source for example samples from three different mice of the same strain or separate extractions of the same cell line or tissue sample When an experiments uses biological replicate groups in a gene expression study the values displayed in the Biological Replicates tab are calculated by combining the results of the separate biological samples and treating this collection as a single population that is as one sample For AC computations normalizing by the endogenous control in a singleplex experiment the software treats separate biological samples as unpair
25. Multicomponent Contains the spectral data used by the ViiA 7 Software to page 205 data generate the multicomponent plot that displays the contribution of each dye over the duration of the PCR run Results data Contains the normalized processed and analyzed data page 206 generated by the ViiA 7 Software Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security File header Appendix E ViiA 7 Software Reference Export formats and file specifications The plate setup file begins with a header that describes the qualities of the ViiA 7 Instrument used to run the experiment and several other general experiment properties Each line starts with an asterisk and ends with a carriage return in the following pattern lt field name gt lt field value gt Note The ViiA 7 Software automatically removes any leading and trailing white space around the field name and field value The header contains the lines listed in the following table Field Description Output Block Type The model of the sample block installed to the ViiA 7 Instrument at the time the experiment was run 96 384 well Of array card Calibration Expired Expiration status of the calibration Indicates whether the calibration of the ViiA 7 Instrument was current at the time that the experiment was run Yes Or No Chemistry The chemistry of t
26. Purge Login Success Failure Logout Success Run Start Stop Completed Failed Aborted Error User Account Create Update Select the System Configuration History tab Display the records of interest Filter the list to decrease the time required to generate reports IMPORTANT You cannot cancel a report after you click a view button Click View Report Inthe Report screen click tool bar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item To print the report click d Print e To save the report electronically PDF click H Save Close the report Archive purge and The audit archive function makes a copy of audit records Purge makes a copy of audit restore audit records records and then deletes them You can use the Restore function to restore purged audit records Archive and purge To selectively archive or purge delete system configuration or action audit records 1 2 3 4 Select the System Configuration History tab Select records in the appropriate screen Click Archive or Purge If you select Archive specify a location and name for the archive file asz Restore To restore system configuration or action audit records click Restore then select the ASZ file to restore Applied Biosystems ViiA 7 Real Time PCR System User Guide 113 Calibration Maintenance Networking and Security 4 Chapter 4 Sec
27. Roles All user accounts with Active status and all user roles and associated permissions in case a user account specifies a user role that does not exist on the system into which you import the profiles System amp Roles Contains all system settings and all user roles and associated permissions 3 Click Export or OK 4 When prompted specify the name and location for the exported file dat then click Save A message is displayed when the export completes Import 1 In any screen in the SAE module click Import in the navigation pane 2 Select the dat file to import then click Open A message is displayed asking if you want to overwrite the current system configuration Click Yes If any imported user accounts already exist on the system you are prompted to overwrite or skip each account 120 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 4 2 SECTION 4 2 Section 4 2 Users Users overview Security Log in Permissions The Security Audit and Electronic Signature SAE module is an optional component of the ViiA 7 Software The module provides the following functionality e System security Controls user access to the software e Auditing Tracks changes made to library items actions performed by users and changes to the Security Audit and Electronic Signature settings e Electronic signature Requires users
28. see Plate setup file format on page 192 File structure Sample file header row The sample file begins with an optional header row that contains column headers for well number Well sample name Sample Name and optional custom properties names The order of the columns is important and cannot be changed Sample file body A body of rows containing the sample data follows the optional header row Each body row defines the sample information for a single well on the consumable including well number sample name and any applicable custom fields The body can contain data for a subset of wells on the consumable so the rows for empty wells can be omitted from the file The sample body rows can occur in any order column Description Valid values name Well The number of the well on the consumable lt Positive integer 1 to 96 where the well numbers start at 1 for well A1 384 gt upper left corner and increase from left to right and from top to bottom Sample The name of the sample contained by the lt 100 character string gt Name associated well Custom1 Optional Additional text that describes the lt 1024 character string gt Custom6 sample in the well Example file Well Sample Name Customl Custom2 Custom3 Custom4 Custom5 Custom6 21 Sample ltestl test2 test3 test4 test5 test6 22 Sample 2testl test2 test3 test4 test5 test 23 Sample 3testl test3 test4 test5 test6 1
29. 0 F Fuse cover Dual 12 5A Time Lag T 250VAC 5 x 20 mm electrical fuses that protect the ViiA 7 Instrument from excessive electrical current G Power port The 100 240VAC port that provides power to the ViiA 7 Instrument Use the Ethernet cable supplied with the ViiA 7 System to connect the ViiA 7 Instrument Ethernet 1 port to the network interface card in the computer Applied Biosystems ViiA 7 Real Time PCR System User Guide 25 Calibration Maintenance Networking and Security 1 Chapter 1 Getting Started ViiA 7 System hardware Bar code readers The Applied Biosystems ViiA 7 Real Time PCR System can include two bar code readers for data entry and plate recognition e A hand held bar code reader for scanning plates manually e A fixed position bar code reader for automatically scanning plates as they are loaded into the instrument available only with the Twister II Robot Both bar code readers use 670 nm Class II lasers to scan plates and both readers are capable of reading Code 128 alphanumeric which supports 128 ASCII character bar codes The bar code readers are optional and available depending on the system configuration About the hand held WARNING LASER HAZARD Exposure to direct or reflected laser light can bar code reader burn the retina and leave permanent blind spots Never look into the laser beam Remove jewelry and anything else that can reflect the beam into your eyes Protect o
30. 1 125 Laptop 1 5 90 100 240 10 50 60 Monitor 1 5 65 Twister II Robott 2 5 150 t The Twister II Robot is an optional component of the ViiA 7 System Note The instrument monitor desktop computer Twister II Robot and laptop computer self adjust for 100v 240v input voltages of 50 60 Hz Environmental requirements Requirement Description Altitude Less than 2000 m 6500 ft above sea level Temperature 15 to 30 C 59 to 86 F Do not place the ViiA 7 Instrument next to heaters cooling ducts or in direct sunlight Temperature fluctuations can affect performance Humidity 20 to 80 relative humidity noncondensing Pollution Pollution Degree rating of 2 Location For indoor use only IMPORTANT Do not locate the ViiA 7 Instrument next to e Vibration sources such as a centrifuge pump or compressor Excessive vibration affects instrument performance e Electrically noisy devices such as a refrigeration unit t The ViiA 7 Instrument can be used in an environment that contains nonconductive pollutants only dust particles or wood chips Typical environments with a Pollution Degree 2 rating are laboratories sales and commercial areas 22 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security ViiA 7 System layout and connections Chapter 1 Getting Started Specifications and layout The ViiA 7 System co
31. 1 NTC Port 2 Unknown A Port 3 Unknown B Port 4 Standard 200 Copies Port 5 Standard 400 Copies Port 6 Standard 800 Copies Port 7 Standard 1600 Copies Port 8 Standard 3200 Copies 44322654 15 to 25 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Consumables Appendix D Parts and Materials Consumables Note For consumable shelf life expiration date see the package label The following consumables are to be used with the Applied Biosystems ViiA 7 Real Time PCR System ViiA 7 System consumable Pale Applied Biosystems Array Card Staker Sealer 4331770 Array Card Bucket Clip Set 1st Generation 4337762 2nd Generation 4442571 Array Cards 8 Port Empty Empty Array Card Kit 4 pk 4334812 Empty Array Card Kit 4351471 Centrifuge Buckets Array Card 1st Generation 4337230 2nd Generation 4442573 Clip Array Card Centrifuge Adapter 4334682 MicroAmp Fast 8 Tube Strip 0 1 mL 125 strips 4358293 MicroAmp Fast Optical 96 Well Reaction Plate 10 plates 4346906 with Bar Code 0 1 mL 200 plates 4366932 MicroAmp Optical 96 Well Reaction Plate 0 2 10 plates N8010560 mL 500 plates 4316813 MicroAmp Optical 96 Well Reaction Plate with 10 plates 4306737 PAE p 500 plates 4326659 MicroAmp Optical 384 Well Reaction Plate 1000 plates 4343370 MicroAmp Optical 384 Well
32. 11 Review the standard curve a Select the Standard Curve tab b Click the upper left corner of the Plate Layout to select all wells c Verify that the R2 value is greater than or equal to 0 990 If the R2 value is less than 0 990 repeat the RNase P experiment using a different RNase P plate If the problem persists contact Applied Biosystems 12 In the Analysis screen click Next remove the plate or array card when the ViiA 7 Instrument ejects the tray arm then discard the plate or array card WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can be heated to 100 C Before removing the plate be sure to wait until it reaches room temperature IMPORTANT If the ViiA 7 Instrument does not eject the plate remove the plate as explained in Troubleshoot the RNase P experiment on page 83 13 Click Finish then click Yes when prompted to save the experiment 82 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Troubleshoot the RNase P experiment Chapter 2 Calibration and Maintenance Verify the instrument performance Problem symptom Possible cause Action More than the maximum number of outliers are present in RNase P data Possible contamination Pipetting inaccuracy Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array ca
33. 2 Calibration and Maintenance Verify the instrument performance Problem symptom Possible cause Action Well displays the NOSIGNAL flag indicating the well produced very low or no fluorescence signal Missing reaction mix resulting from pipetting error If a well is flagged confirm the results 1 Consider omitting the well from the analysis 2 Note the location for each flagged well and check each corresponding well in the reaction plate for evaporation or low reaction volume 3 Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Well displays the OFFSCALE flag indicating the fluorescence signal for one or more dyes in the well exceeds the instrument s maximum detectable range for one or more cycles e Fluorescent contaminant on the reaction plate or sample block e Fluorescent contaminant in the reaction 1 Perform a background calibration If you detect fluorescent contamination decontaminate the sample block 2 Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Well displays the OUTLIERRG flag indicating the C of the well deviates significantly from C values in the associated replicate group
34. 25 0 0 SNP rs15934 RGB 0 75 0 UNKNOWN G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 12 Sample01 RGB 25 0 0 SNP rs15934 RGB 0 75 0 UNKNOWN G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 13 Sample02 RGB 25 0 0 SNP rs15934 RGB 0 75 0 UNKNOWN G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 14 Sample02 RGB 25 0 0 SNP rs15934 RGB 0 75 0 UNKNOWN G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 13 Sample02 RGB 25 0 0 SNP rs15934 RGB 0 75 0 UNKNOWN G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB suoneoyinads pue s ewsoj jJOdW aduaajay JEMYOS VIIA 3 xipuaddy Appendix E ViiA 7 Software Reference E Import formats and file specifications Sample file format The ViiA 7 Software can import sample files to populate sample information into an open experiment A sample file is a tab delimited ASCII text file txt that contains sample well designations and custom sample properties The files can be created manually using a text processor or generated automatically by third party applications IMPORTANT To guarantee successful import the file must contain all the elements described in the following section and in the order that they appear Note The command line application see page 184 does not import sample files If you are using the application to create experiments use plate setup files to import sample information into the new experiments
35. 4 Touch Save to save the settings then touch OK when prompted 5 Touch to return to the Main Menu Applied Biosystems ViiA 7 Real Time PCR System User Guide 153 Calibration Maintenance Networking and Security A Appendix A Manual Instrument Operation Administrate the instrument from the touchscreen Define the network settings 1 If the instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press Ot In the Main Menu touch Settings then touch Set Network Information Note The Set Network Information screen displays the Media Access Control MAC address of the ViiA 7 Instrument below the Autodiscovery and Smart Monitoring check boxes The MAC address can be used to uniquely identify the ViiA 7 Instrument on the network TM Touch Autodiscovery to make the ViiA 7 Instrument discoverable by computers that are running the ViiA 7 Software Touch Smart Monitoring to enable the feature on the ViiA 7 Instrument The Smart Monitoring feature allows Applied Biosystems service personnel to monitor the status of the ViiA 7 Instrument remotely through an internet connection Smart Monitoring employs multiple layers of security including a Secure Sockets Layer SSL and Lightweight Directory Access Protocol LDAP authentication to provide real time troubleshooting and problem resolution for the ViiA 7 Instrument For a detailed description of the Smart Monitoring Service see the S
36. 6 c cece eee ees 160 m Power on the ViiA 7 System 2 eee eens 160 m Power off the ViiA 7 System 0 2 eens 161 BM Store the VisA 7 Systemic iiss ein sie manem bare wile diese a aa a Siea h a ESAS 162 E Move the ViiA 7 System esi nies sinak nane eee ens 163 Applied Biosystems ViiA 7 Real Time PCR System User Guide 159 Calibration Maintenance Networking and Security Appendix B Power On or Off Store and Move the ViiA 7 System Place the ViiA 7 System on standby Place the ViiA 7 System on standby If left unattended the ViiA 7 Instrument automatically enters standby mode to conserve power To enter standby mode manually touch on the ViiA 7 Instrument touchscreen Power on the ViiA 7 System To power on the ViiA 7 System from a powered off state 1 Toggle the power button on the rear of the ViiA 7 Instrument then wait for it to boot Note The ViiA 7 Instrument is ready to use when the touchscreen displays the Main Menu 2 If you have an Applied Biosystems Twister II Robot toggle the power button on the rear of the Twister II Robot Note The Twister II Robot is ready to use when the power LED illuminates 3 Power on the monitor 4 Power on the ViiA 7 System computer a Press the power button of the computer then wait for it to boot b When the Login screen appears enter your user name and password then click OK c In the desktop double click ViiA 7
37. 6 Install the new lamp into the ViiA 7 Instrument a Slide the lamp release lever upward b Firmly grasp the lamp place it into the slotted mount then carefully slide the lamp downward into place 7 Close the access door 8 Plug in and power on the ViiA 7 Instrument IMPORTANT Confirm that the access door is completely closed The ViiA 7 Software displays an error message if the door is not completely closed and latched or if the lamp is not seated correctly 9 Start the ViiA 7 Software then perform an ROI calibration While the ViiA 7 Instrument is running look through grating of the access door and verify that the lamp is illuminated e Ifthe lamp is illuminated the lamp has been installed successfully e Ifthe lamp is not illuminated the replacement halogen lamp may be defective Replace the lamp again If the second lamp does not illuminate check the ViiA 7 Instrument fuses for failure see page 130 10 After replacing the lamp perform the following calibrations in the specified order a Background calibration b Uniformity calibration c Dye calibration d Normalization calibration Applied Biosystems ViiA 7 Real Time PCR System User Guide 129 Calibration Maintenance Networking and Security 5 Chapter5 Service Replace the instrument fuses Replace the instrument fuses Materials required Replace the fuses 130 Replace the ViiA 7 System fuses when the fuses
38. 64 5 25 4 60 7 133 5 Computer Laptop 35 8 14 1 25 7 10 1 35 8 14 1 2 6 5 7 Desktop 18 7 7 3 44 5 17 5 41 0 16 1 10 9 24 0 Monitor 44 7 17 5 19 3 7 6 36 6 14 4 6 9 15 2 Keyboard 44 7 17 5 15 25 6 0 5 0 2 0 0 1 0 2 Total footprint 233 91 7 86 33 8 79 7 31 4 77 9 171 5 Weight varies depending on the sample block installed Computer properties differ depending on the computer ordered with the ViiA 7 System laptop or desktop 20 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security ViiA 7 System with Twister IlI Robot dimensions 15 24 cm 6 in 53 3 cm 21 in 213 36 cm 84 in Chapter 1 Getting Started Specifications and layout ul GZ Wo g g9 ul ze wo BZ LE o S o Q 3 g 5 Width Depth Height Weight Component cm in cm in cm in kg lbs Instrument 53 3 21 0 63 5 25 0 64 5 25 4 60 7 133 5 Computer Laptop 35 8 14 1 25 7 10 1 35 8 14 1 2 6 5 7 Desktop 18 7 7 3 44 5 17 5 41 0 16 1 10 9 24 0 Monitor 44 7 17 5 19 3 7 6 36 6 14 4 6 9 15 2 Keyboard 44 7 17 5 15 25 6 0 5 0 2 0 0 1 0 2 Twister II Robot 53 3 21 0 77 0 28 0 97 0 38 0 31 8 70 0 Total footprint 311 0 122 2 86 0 33 9 97 0 38 0 109 7 241 5 Weight varies depending on the sample block installed Computer specification differs depending on th
39. 7 Software licenses on page 133 Update the ViiA 7 Instrument firmware You can use the ViiA 7 Instrument touchscreen to update the ViiA 7 Instrument firmware See Update the instrument firmware on page 150 for more information 132 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter5 Service 5 Manage ViiA 7 Software licenses Manage ViiA 7 Software licenses You can use the License Central feature to monitor activate or install the licenses that control access to the ViiA 7 Software base application and associated modules About ViiA 7 Software license keys and files The ViiA 7 Software and associated modules require the installation and maintenance of valid license files for continued operation The license files are generated by the Applied Biosystems website when a license key is activated Each file pairs a software license key with the computer from which the key was activated After a key is activated and a license file is generated the file cannot be transferred to another computer To transfer a license between computers you must reactivate the license key using the ViiA 7 Software on the target computer Note ViiA 7 Software licenses are valid for a limited time and they must be renewed regularly If a license has expired or is nearing expiry the ViiA 7 Software displays a warning when the software is started Not
40. Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix B Power On or Off Store and Move the ViiA 7 System Move the ViiA 7 System Move the ViiA 7 System Perform this procedure to safely move the ViiA 7 System short distances for example between laboratories of the same building CAUTION PHYSICAL INJURY HAZARD Do not attempt to lift the ViiA 7 Instrument or any other heavy objects unless you have received related training Incorrect lifting can cause painful and sometimes permanent back injury Use proper lifting techniques when lifting or moving the ViiA 7 Instrument At least two people are required to lift it IMPORTANT Moving your ViiA 7 System can create subtle changes in the alignment of the instrument optics Recalibrate the instrument if necessary Materials required None How to handle the sample block and heated cover To prevent damaging or contaminating the sample block or the heated cover handle the assemblies as shown below After you remove each assembly from the ViiA 7 Instrument place them on a clean dry surface or in its shipping container Prepare for the ViiA 7 System components 1 Power off the ViiA 7 Instrument and computer 2 When the ViiA 7 System and computer are powered off disconnect all ViiA 7 System components and package the cabling for the move 3 Prepare the ViiA 7 Instrument
41. Calibration Maintenance Networking and Security 210 Appendix E ViiA 7 Software Reference Export formats and file specifications Genotyping The following table describes the results data exported from genotyping experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Sample Name The name of the sample contained by the well 100 character string SNP Assay The name of the SNP assay added to the well 100 character string Name Task The task assigned to the target in the well UNKNOWN or NTC Allele1 Rn The raw fluorescence associated with the allele 1 Float probe of the SNP assay at the well normalized to the fluorescence of the passive reference dye Allele2 Rn The raw fluorescence associated with the allele 2 Float probe of the SNP assay at the well normalized to the fluorescence of the passive reference dye Pass Ref The raw fluorescence of the passive reference at Float the well Qualityl The confidence of the automatic allele call Float 1 to 100 Call The allele call assigned to the sample at the Homozygous specified well lt allele x allele x gt Heterozygous lt allele x allele y gt or Negative Control NC Method The method used to call alleles Auto or Manual Allele Whe
42. Connect the ViiA 7 Instrument to the network 00 c cece cence nee ees 91 Materials required 020 c cece ect erener 91 Define the ViiA 7 Instrument internet protocol settings 0 c eee eee 91 Connect the computer to the network 000cc eee eee eee eee eee 92 Materials required nnna cece tcc e eee eens 92 Computer requirement 00 00 cece eect eeee 92 Collect required information 00cce cece eect teeees 92 Setup the computers seen Senedd acne ete od iain nde oe Meal ee ceive tel E 92 Install the ViiA 7 Software 2 0 0 0 ccc cee cece eee ence eens 93 Monitor the ViiA 7 Instrument 0 000 cece eee e rrr errre 94 About remote monitoring 22000 eee eee eee eee eens 94 Monitor the status of ViiA 7 Instrument during a rUn 0 ccc cence eee 94 Upload or download an experiment or template to a ViiA 7 Instrument 95 Enable or change the calibration reminders 220ee cece cece ee eeeee 96 Applied Biosystems ViiA 7 Real Time PCR System User Guide 7 Calibration Maintenance Networking and Security Contents CHAPTER 4 Security Audit and Electronic Signature 2000008 99 Section 4 1 Administrators 0 ccc cece eee eee 101 Administrators overview 2 Sessea irra aeaii aN ENR enna 101 Example applications serioen edena a aaa eased a ee Ade 102 Configure the security
43. E Appendix E ViiA 7 Software Reference Export formats and file specifications Standard Curve Relative Standard Curve and Comparative Cy experiments The following table describes the results data exported from standard curve relative standard curve and comparative Cy experiments Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 t Sample Name The name of the sample contained by the well 100 character string Detector The name of the target assay added to the well 100 character string Name Reporter The reporter dye that labels the probe for the 100 character string target assay Task The task assigned to the target in the well UNKNOWN NTC or STANDARD CT The calculated threshold cycle C7 for the target Float at the specified well Quantity e Unknown wells The calculated quantity for Float the sample at the well e Standard wells The quantity assigned to the standard at the well Quantity Mean e Unknown wells The average quantity of the Float replicate wells for the target sample e Standard wells The quantity assigned to the replicate wells for the target sample Quantity SD The standard deviation of the average quantity of Float the replicate wells for the target sample combination Ct Median The median Cy of the replicate wells for the Float specified target sample combination Ct Mean The average Cy of the
44. In standard curve experiments the quantity of target in the standard In the software the units for standard quantity can be for mass copy number viral load or other units for measuring the quantity of target Standard quantity can also refer to dilution factor When defining a standard curve in the software the highest quantity A component of the thermal profile For each step in the thermal profile you can set the ramp rate ramp increment for melt curve steps hold temperature and hold time duration You can turn data collection on or off for the ramp or the hold parts of the step For cycling stages a step is also defined by the AutoDelta status PCR reaction components that consist of two primers designed to amplify the target and SYBR Green dye to facilitate detection of the PCR product Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security system dye TaqMan reagents target target color task technical replicates temperature plot template template file thermal profile Glossary Dye supplied by Applied Biosystems and precalibrated on the ViiA 7 System Before you use system dyes in your experiments make sure the system dye calibration is current in the Instrument Console The system dyes are e FAM dye e JOE dye e ROX dye e NED dye e SYBR Green dye e TAMRA dye e VIC dye PCR reaction components that consist
45. In the Home screen of the ViiA 7 Software click Instrument Console b In the Instrument Console select your ViiA 7 Instrument then click Add to My Instruments Select the ViiA 7 Instrument then click Manage Instrument In the Instrument Manager click Maintenance then click ROI In the ROI Calibration screen click Start Calibration In the ROI dialog box click Next until prompted to load the ViiA 7 Instrument When the side door opens load the sealed plate Ensure that the plate array card is properly aligned in the holder g In the ROI dialog box select Check the box when the ROI calibration plate has been loaded click Next twice then click START RUN to start the calibration hoan 3 When the run is complete inspect the ROI images a Select the first filter from the Filter drop down list b Record the coordinate of the well that contains the lowest concentration of dye and that is encircled by a ring This well contains the optimal concentration of the custom dye at the given filter c Repeat steps 3a and 3b for the remaining filters d After you determine the optimum concentration for each filter determine the optimum concentration for the custom dye Compare the results from all filters then select the concentration that yields the highest possible signal in all filters 4 Discard the plate WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can be heated to 100 C
46. Max The maximum relative quantity calculated for the replicate wells of the target sample combination The upper limit of the confidence interval Float Ct Mean The average C of the replicate wells for the specified target sample combination Float Delta Ct Mean The average AC of the replicate wells for the specified target sample combination Float Delta Ct SD The standard deviation of the AC for the replicate well Depending on the analysis settings this column may by replaced with Delta Ct SE the standard error of the AC Float Delta Delta Ct The AAC value of the replicate wells for the specified target sample combination Float Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference Export formats and file specifications The following table describes the results data exported from high resolution melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below BioGroup analysis results study Column Description Output Biogroup The name of the biological replicate group 100 character string Name Target The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN or NTC Omitted Indicate
47. PCR experiment The amplification data R are the raw fluorescence readings collected by the ViiA 7 Instrument normalized to the fluorescence from the passive reference If available the exported amplification data also exports the baseline compensated normalized fluorescence data AR calculated by the ViiA 7 Software The section begins with a column header followed by the amplification data where each row contains the data for a single well separated by tab characters If a well contains more than one assay target the ViiA 7 Software lists the data for each additional assay on separate rows repeating the well number and sample information Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Cycle The cycle of the run during which the ViiA 7 Integer Instrument recorded the fluorescence Target Name Genotyping experiments The name of the SNP assay assigned to the well and the allele name lt SNP assay name gt lt allele name gt All other experiments The name of the target assigned to the well Name of the target Rn The raw fluorescence for the associated well Float normalized to the fluorescence of the passive reference dye reporter signal or passive reference signal Delta Rn The baseline compensated R value for the Float associated well Well numbers start at 1 for well A1 upper left corner and increase from left t
48. PN 4442663 Explains how to integrate a robotic plate handler with the ViiA 7 System Intended for engineering personnel who are responsible for integrating a robotic plate handler with the ViiA 7 System Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 237 Documentation and Support Related documentation 238 Document Purpose and audience ViiA 7 Software v1 Help Explains how to use the ViiA 7 Software to Set up run and analyze experiments using the ViiA 7 System Monitor a networked ViiA 7 System Calibrate a ViiA 7 System Perform an RNase P run Intended for laboratory staff who perform experiments using the ViiA 7 System and who are responsible for the maintenance of the ViiA 7 System Portable document format PDF versions of this guide and other ViiA 7 Instrument Documentation are also available on the ViiA 7 Software CD Note To open the user documentation included on the Applied Biosystems ViiA 7 Real Time PCR Instrument Software CD use the Adobe Acrobat Reader software available from www adobe com Note For additional documentation see Obtaining support on page 240 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Documentation and Support Obtaining information from the Help system Obtaining
49. Reaction Plate with 1000 plates 4343814 Pal Voge 500 plates 4326270 50 plates 4309849 MicroAmp Optical 8 Cap Strip 300 strips 4323032 MicroAmp Optical 8 Tube Strip 0 2 mL 1000 tubes 4316567 MicroAmp Optical Adhesive Film 4311971 Replacement Lamp for OptiFlex System 4443978 181 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix D Parts and Materials Consumables 182 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security APPENDIX E ViiA 7 Software Reference This appendix covers m ViiA 7 Software command line application 6 6 cece eee eee 184 m Import formats and file specifications 6 000 c cece eens 191 m Export formats and file specifications s 0 6 ccc cece eee eee 199 Applied Biosystems ViiA 7 Real Time PCR System User Guide 183 Calibration Maintenance Networking and Security E Appendix E ViiA 7 Software Reference ViiA 7 Software command line application ViiA 7 Software command line application The ViiA 7 Software includes a command line application that allows you to generate and export batches of experiment files from an MS DOS prompt or a batch file The application is intended for advanced users who choose to create or export experiments using a scripting language IMPORTANT After you use the command line applicati
50. Real Time PCR System User Guide 29 Calibration Maintenance Networking and Security Chapter 1 Getting Started ViiA 7 System software ViiA 7 System software The ViiA 7 System includes a suite of software applications that can be used to calibrate run automate and integrate the ViiA 7 System into a laboratory workflow The basic installation of the ViiA 7 Software contains the components described below however additional software may be available for the ViiA 7 System Visit the ViiA 7 System website for a complete list of compatible software www appliedbiosystems com viia7 Note Visit the ViiA 7 System website for updates and patches for the ViiA 7 Software and ViiA 7 Instrument Firmware Computer requirements The requirements for the computer used to operate the ViiA 7 Instrument can vary depending on the version of the ViiA 7 Software that you are running To determine the computer requirements for your ViiA 7 System check the ViiA 7 Software release notes at the following location D AppliedBiosystems ViiA7 Software release notes html Software installation 30 The default installation of the ViiA 7 System partitions the computer hard drive to create the logical drives shown below Drive Software Description C Microsoft Windows OS Operating system files D ViiA 7 Software Used to calibrate and perform experiments on the ViiA 7 Instrument
51. Real Time PCR System User Guide 41 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the ROI calibration Perform the ROI calibration Materials required 96 Well Plate Sample Block 384 Well Plate Sample Block Array Card Sample Block A regions of interest ROI calibration maps the positions of the wells on the sample block of the ViiA 7 Instrument The ViiA 7 Software uses the ROI calibration data to associate increases in fluorescence during a run with specific wells on the plate The ViiA 7 Instrument uses a set of optical filters to distinguish the fluorescence emissions gathered during runs You must generate a calibration image for each individual filter to account for minor differences in the optical path e 96 Well Region of Interest ROI and Background Plates e Centrifuge with plate adapter e Powder free gloves e Safety goggles Note Only the ROI plate is required for this calibration e 384 Well Region of Interest ROI and Background Plates e Centrifuge with plate adapter e Powder free gloves e Safety goggles Note Only the ROI plate is required for this calibration e Applied Biosystems Array Card filled with ROI Calibration Mix e Applied Biosystems Array Card Staker Sealer e Centrifuge with centrifuge array card carrier clips and buckets e Powder free gloves e Safety goggles e Pipettor 200 uL with pipette tips When to perform the calibrati
52. Sample 5testl test2 test3 test4 test5 test6 2 Sample 6test1 test2 test3 test4 test5 test 3 Sample Ttestl test2 test3 test4 test5 test6 4 Sample 8testl test2 test3 test4 test5 test Applied Biosystems ViiA 7 Real Time PCR System User Guide 197 Calibration Maintenance Networking and Security E Appendix E ViiA 7 Software Reference Import formats and file specifications Bar code file format File structure Example file The ViiA 7 Software command line application can import bar code files to populate experiment files eds it generates with bar code information A bar code file is a tab delimited ASCII text file txt that contains a list of bar codes The files can be created manually using a text processor or generated automatically by third party applications IMPORTANT To guarantee successful import the file must contain all the elements described in the following section and in the order that they appear The bar code file contains a list of bar codes where each line defines a single bar code terminated by a carriage return The bar codes can occur in any order and cannot contain starting or trailing white space Note The ViiA 7 Software command line application does not validate the bar codes HA996346102 1B894812348 DD834814679 EK209825848 AF092387348 FF225676243 Assay information file 198 The ViiA 7 Software command line application can import data for Applied Biosystems assays
53. TaqMan RNase P Fast 384 Well Instrument Verification Plate Array Card Sample e Applied Biosystems Array Card Staker Sealer Block e Centrifuge with centrifuge array card carrier clips and buckets e Powder free gloves e Safety goggles e Pipettor 200 uL with pipette tips e TaqMan RNase P Array Card Instrument Verification Reagents Kit Applied Biosystems Array Card TaqMan RNase P Array Card Instrument Verification Reagents Kit including tubes with reagent mix for each port 8 tubes total When to perform the RNase P instrument verification experiment We recommend performing an RNase P instrument verification experiment e After moving the ViiA 7 Instrument to another location e As needed to verify the function of the ViiA 7 System 74 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Verify the instrument performance About the RNase P kits The instrument verification experiment uses one of two instrument verification kits available from Applied Biosystems The kits differ only in the consumable format for which they are designed a TaqMan RNase P Instrument Verification Plate for ViiA 7 Instruments with 96 384 well sample blocks and an Array Card RNase P Kit for ViiA 7 Instruments with array card sample blocks About the TaqgMan The RNase P plate is preloaded with the reagents necessary for the detect
54. Time PCR System The following materials are required to calibrate the ViiA 7 System M 384 well sample block kits 0 0 0 6 see below mM 96 well sample block kits 0 06 eee eens 178 m Fast 96 well sample block kits 0 6 ices 179 m Array card sample block kits 6 ee ene 180 Note For reagent or consumable shelf life expiration date see the package label 384 well sample block kits ViiA 7 System consumable Pisin iog 384 Well Spectral Calibration Plate with FAM Dye 4432271 15 to 25 384 Well Spectral Calibration Plate with VIC Dye 4432278 384 Well Spectral Calibration Plate with ROX Dye 4432284 384 Well Spectral Calibration Plate with NED Dye 4432302 384 Well Spectral Calibration Plate with SYBR Green Dye 4432290 384 Well Spectral Calibration Plate with TAMRA Dye 4432296 384 Well Region of Interest ROI and Background Plates 4432320 e 384 Well Region of Interest ROI Calibration Plate e 384 Well Background Plate 384 Well Normalization Plates with FAM ROX and VIC ROX 4432308 Dyes e 384 Well Normalization Plate with FAM ROX Dye e 384 Well Normalization Plate with VIC ROX Dye Kit TaqMan RNase P Fast 384 Well Instrument Verification Plate 4455280 e 384 Well TagqMan RNase P Fast Instrument Verification Plate Applied Biosystems ViiA 7 Real Time PCR System User Guide 177 Calibration Maintenance Networking and Securit
55. Version field in the Instrument Properties pane The installation is successful if the ViiA 7 Instrument powers on and if the Heated Cover Firmware Version field displays a version number Perform the following calibrations in the specified order ROI calibration Background calibration Uniformity calibration Dye calibration then Normalization calibration Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 5 Service 5 Replace the plate adapter Replace the plate adapter Replace the plate adapter in the event of a hardware failure or if you want to change the consumable format of the ViiA 7 Instrument WARNING PHYSICAL INJURY HAZARD During instrument operation the sample block and heated cover can be heated to 100 C Before removing the heated cover be sure to wait until it reaches room temperature Materials required e Safety glasses e Powder free gloves e Plate adapter Replace the plate adapter IMPORTANT If you are installing a plate adapter of a different format you may also be required to change the sample block to match the new consumable format 1 Touch the instrument touchscreen to awaken it then press 2 In the Main Menu touch g 3 When the tray arm opens pull the latch then lift and remove the plate adapter 4 Attach the new adapter to the tray arm then pull the latch to allow the adapter to lower into place
56. ViiA 7 Instruments within a study Materials required 96 WellL Plate e 96 Well Normalization Plates with FAM ROX and VIC ROX Dyes Sample Block e Centrifuge with plate adapter e Powder free gloves e Safety goggles 384 Well Plate e 384 Well Normalization Plates with FAM ROX and VIC ROX Dyes Sample Block e Centrifuge with plate adapter e Powder free gloves e Safety goggles Array Card Sample e Applied Biosystems Array Cards filled with Block FAM ROX dye mix VIC ROX dye mix e Applied Biosystems Array Card Staker Sealer e Centrifuge with centrifuge array card carrier clips and buckets e Powder free gloves e Safety goggles e Pipettor 200 uL with pipette tips When to perform the calibration Perform a normalization calibration every 6 months or as often as necessary depending on instrument use About the normalization calibration data During the normalization calibration the ViiA 7 Software captures a series of images of each normalization plate using each instrument filter The normalization calibration yields a Pass or Fail result for each normalization plate used Applied Biosystems ViiA 7 Real Time PCR System User Guide 69 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the normalization calibration Prepare the calibration plate or array card Prepare the normalization plates 70 IMPORTANT Wear
57. ViiA 7 Passive Reference ROX Sample Setup Well Sample Name Sample Color SNP Assay Name SNP Assay Color Task Allelel Name Allelel Color Allelel Reporter Allelel Quencher Allele2 Name Allele2 Color Allele2 Reporter Allele2 Quencher Comments 1 Neg Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 NTC G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 2 Neg Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 NTC G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 3 Neg Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 NTC G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 4 All Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 PC_ALLELE_1 G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 5 All Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 PC_ALLELE_1 G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 6 All Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 PC_ALLELE_1 G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB a Al2 Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 PC_ALLELE_2 G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 8 Al2 Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 PC_ALLELE_2 G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 9 Al2 Control RGB 25 0 0 SNP rs15934 RGB 0 75 0 PC_ALLELE_2 G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 10 Sample01 RGB 25 0 0 SNP rs15934 RGB 0 75 0 UNKNOWN G RGB 0 0 50 VIC NFQ MGB A RGB 0 50 0 FAM NFQ MGB 11 Sample01 RGB
58. When a message is displayed by the Windows operating system instructing you to defragment For more information on maintaining the hard drives see the Windows Operating System Help then search the Help to find information on the Disk Cleanup and Disk Defragment utilities IMPORTANT Do not run the disk management utilities and ViiA 7 Software at the same time Archive and back up experiment files Archive experiment To conserve space on the computer hard drive older EDS files can be archived using a files regularly data compression utility Several commercial compression utilities are available to store experiment files in the ZIP or ARC archive format Back up experiment We strongly recommend that you back up your experiments Backing up data files e Protects against potential loss of data caused by an unforeseen failure of the computer or its hard drive s e Conserves space on the hard drive and optimizes performance Develop a data We recommend developing a strategy for managing the files produced by the ViiA 7 management Software strategy Note Real time runs generate significantly more data than genotyping or presence absence experiments During one day of real time operation the ViiA 7 System can generate more than 10 MB of data Check disk space If you perform real time experiments on your ViiA 7 System check the amount of available space on your hard drive weekly When the hard drive is within 20 of
59. Yes RNaseP Perform an RNase P experiment Yes No Yes Instrument Add or remove ViiA 7 Instrument from No No Yes Configuration monitoring Security Configure the security and audit feature No No Yes Ci Log into user sessions that have timed out No No Yes Perform E Signing Yes Yes Yes 108 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators 4 Manage user accounts Edit a user role 1 In the Roles screen select a user role then click Edit 2 Edit settings as needed You cannot edit the Administrator user role 3 Click Save Role View and print a user report 1 In the ViiA 7 Software select Tools gt Security gt Settings 2 In the Security Settings dialog box select the Users or Roles tab 3 Click View Report 4 In the Report screen click tool bar options to manipulate the report as needed Place the mouse pointer over an item for a description of the item 5 Click eh Print to print the report or click ei Save to save the report electronically PDF Close the report Applied Biosystems ViiA 7 Real Time PCR System User Guide 109 Calibration Maintenance Networking and Security 4 Chapter 4 Security Audit and Electronic Signature Manage auditing Manage auditing Access the Audit screen and enable or disable auditing Use the Audit screen to control the auditing state enabled disabled the eve
60. account setup and security policies Note Security policies apply to all user accounts 1 2 7 In the ViiA 7 Software select Tools gt Security gt Settings In Account Setup specify User Name limits IMPORTANT The software allows spaces in user names Use spaces in user names with caution For information see Spaces in user names and or passwords on page 104 Specify User Password limits a Specify the passwords length limits b Specify password reuse You cannot disable the password reuse restriction c Specify the allowed characters in user passwords spaces and alphabetical numeric uppercase lowercase and special characters commas periods semicolons dashes underscores and tildes In Security Policies specify Password Expiration Account Suspension and Session Timeout settings Note A session times out while a run is in progress if the time out period is exceeded and there is no other user activity In the Open Non Secure Data option select Yes or No to determine whether users can open experiments and templates that were created without security settings Click Set Up Messaging Notification Settings to specify when and how the ViiA 7 Software notifies the administrator of certain security events For information see Set up messaging notifications on page 105 Click Apply Settings The new settings are applied to the user account the next time that the user logs in
61. all experiments unknown samples and dilution series of template such as cDNA are amplified Following a run the instrument software interpolates relative quantities for each unknown sample from the appropriate dilution curve then normalizes the data for each sample or set of replicates as follows target QAvg endogenous control QAvg A user defined biological grouping A replicate group may be a set of identical reactions in an experiment Total number of identical reactions containing identical components and identical volumes A fluorescent dye used to detect amplification With TaqMan reagents the reporter dye is attached to the 5 end With SYBR Green reagents the reporter dye is SYBR Green dye SYBR and HRM specific dyes are DNA binding dyes An oligonucleotide that flanks the 3 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target An enzyme that converts RNA to cDNA See normalized reporter Rn A dye supplied by Applied Biosystems and precalibrated on the instrument ROX dye is used as the passive reference See refSNP ID Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security run method sample sample definition file security auditing and eSignature serial factor slope stage standard Glossary Definition of the reaction volume and the thermal profile
62. any comments you have in the Comments field click Finish then click Yes when prompted to save the calibration results Failed Create another custom dye plate using the next dye concentration greater than the concentration determined in Determine optimum dye concentration on page 168 then perform the calibration again Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security APPENDIX D Parts and Materials This appendix covers B How toorder eirs aces aecee ss etic ea eres oder d BED Re ae es See ha ae 174 MM A CCOSSOPES 3 nicges ce cits Sickie ach Sieh waa eb HSA depo ee E ERIE 176 E Calibration and verification kits 00 cece tenes 177 E Consumables 0 0 0 0c cee tence nent eee nena nees 181 Applied Biosystems ViiA 7 Real Time PCR System User Guide 173 Calibration Maintenance Networking and Security Appendix D Parts and Materials How to order How to order You can order materials and accessories from Applied Biosystems by e Accessing the Applied Biosystems store from the ViiA 7 Software e Ordering directly from the Applied Biosystems store over the internet Note Product availability and pricing may vary according to your region or country Online ordering through the Applied Biosystems Store is not available in all countries Contact your local Applied Biosystems representative for help To order through the website or the ViiA
63. bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in DN a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety To minimize the hazards of chemicals guidelines e Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About SDSs on page 232 e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS e Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as rec
64. character string Name Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN Or NTC RQ The relative quantity calculated for the replicate wells of the target sample combination Float RQ Min The minimum relative quantity calculated for the replicate wells of the target sample combination The lower limit of the confidence interval Float RQ Max The maximum relative quantity calculated for the replicate wells of the target sample combination The upper limit of the confidence interval Float Ct Mean The average Cy of the replicate wells for the specified target sample combination Float Delta Ct Mean The average AC of the replicate wells for the specified target sample combination Float Delta Ct SD The standard deviation of the AC for the replicate well Depending on the analysis settings this column may by replaced with Delta Ct SE the standard error of the AC Float Delta Delta Ct The AAC value of the replicate wells for the specified target sample combination Float Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference E Export formats and file specifications Technical replicate results The following table describes the results data exp
65. contents of the custom text fields found in the Results table of the experiment 1024 character string per field Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom 212 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference Export formats and file specifications Study data The following table describes the results data exported from a study of relative quantification experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Experiment The name of the of the experiment 100 character string Name Well The number of the well on the consumable Integer 1 to 96 384 Omitted Whether the well was omitted from the analysis true or false true or included false Sample The name of the sample contained by the well 100 character string Target The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN or NTC Reporter The reporter dye that labels the probe for the 100 character string target assay Quencher The quencher dye that labels the probe for the 100 character string target assay RQ The relative quantity calculated for the replicate Float w
66. define the ViiA 7 Instrument settings see Define the instrument settings on page 152 enter the instrument name using lower case letters only Network setup workflow 90 Collect the required network information Connect the ViiA 7 Instrument to the network Connect the computer to the network Monitor the ViiA 7 Instrument to test the network connection Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 3 Networking Collect the required network information Collect the required network information Obtain the following information from your network administrator e Network policy for obtaining IP addresses DHCP or static IP IMPORTANT When the ViiA 7 System is set for DHCP APIPA is automatically enabled and the ViiA 7 System will self assign an IP address when no address is supplied by a DHCP server e Ifthe network requires static IP addresses obtain the IP address subnet mask and gateway address for the ViiA 7 Instrument Connect the ViiA 7 Instrument to the network After deciding how to connect the ViiA 7 System to a network set up the ViiA 7 System according to your network policies Materials required Ethernet cable with RJ45 connectors a CAT6 Ethernet cable for a 1000Mbit s network connection or a CAT5 for 100Mbit s connection Define the ViiA 7 Instrument internet protocol settings 1 Use th
67. dye 250 symbols safety 224 system configuration history contents 112 display 111 114 119 system dyes 19 62 251 system shortcuts instrument 155 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Index TAMRA dye 19 62 TaqMan reagents 251 TaqMan RNase P Fast 384 Well Instrument Verification Plate 75 177 target 251 target color 251 task 251 technical replicate 251 technician user role 107 Temperature Plot 251 temperature requirement 22 template 251 template file 251 third party software 31 threshold 252 threshold cycle 252 threshold cycle CT 252 timeout session 104 122 touchscreen instrument 24 142 training information on 240 transfer data to from instrument 95 146 tray arm 24 troubleshoot background calibration 53 dye calibration 68 instrument fuses 130 instrument verification 83 lamp replacement 127 normalization calibration 73 optical calibration 53 RNase P instrument verification experiment 83 ROI calibration 47 sample block decontamination 124 uniformity calibration 59 261 Index U ultraviolet light safety 228 uniformity calibration 55 252 data 55 perform 57 troubleshoot 59 when to perform 55 uninterruptable power supply requirements 29 unknown 252 update firmware 132 150 operating system 131 service packs 131 software 132 UPS requirements 29 USB drive transfer data 145 USB ports 24 25 145 user ac
68. eee eee ee eees 117 Select the actions that require signature 000 cece cece ee ee ees 117 How the software prompts electronic signature cece eee eee eee ee 118 Generate e signature reports 0 00 c eee eee eee eee eee eee ee eeees 119 Display the e sig recordS 0 c cece cece ect teeteeeeeeee 119 Save or print e sig records 2002ce cece eee eee eee eee 119 Save or print the table of e signature events 0000 c cece eee eee eee 119 Export and import user security audit and e signature settings 120 E pO a heath a E a Ie ated ca Wate vee L Gene od es ate eee 120 IDOR ese tesa ten acento ears te eee aes tee eee taal Mae Sanne E NA 120 Section 4 2 USGrs sx cis ceveor cesses cutie weer siete ove a teees 121 USEPS OVERVIEWS iced dt ante ey Pak ea ied ddl ied ede aes 121 Securty gees s eet k bed co rhe seed E seeds 2 A dia ooo ee gad win oe 121 LOGIN wes aaa ie ie Bee a eee ee Sle ee eee Hele wee BEG Sie 121 PerMISSIONS enie ices a toe E Pen bad Maat angele dhe EA 121 Change your password when it expireS 000c cece cece eee eee eeees 121 Account SUSPENSION 000 cece eee eee 122 SESSION tIME OUE nz ey coe gigs eee Sabine eats Cached edt neal oes gal cable ee 122 AUGIty seie a Altech state tet thes Bie is ed Re ete tei ott TAS os 122 Electronic signaturre serisini Sate eae ped di hee Pees ave Ha ae aes 122 Applied Biosystems ViiA 7 R
69. exported from absolute quantification relative quantification or presence absence experiments The body can contain all or some of the data columns below depending on the export configuration Note For genotyping experiments see Genotyping experiments on page 203 Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Sample Name The name of the sample contained by the well 100 character string Sample Color The RGB color of the associated sample RGB lt r gt lt g gt lt b gt 8 Target Name The name of one target in the well if applicable If a well contains multiple targets one row is used per target 100 character string Target Color The RGB color of the associated SNP assay RGB lt r gt lt g gt lt b gt 8 Task The task the target is used for in this well UNKNOWN STANDARD IPC NTC or BlockedIPC Reporter The reporter dye that labels the probe for the 100 character string target assay Quencher The quencher dye that labels the probe for the 100 character string target assay Quantity Standard quantity if applicable This column Float or Integer only appears for Standard Curve and Relative Standard Curve experiments Comments Additional text that describes the well 1024 character string Well numbers start at 1 for well A1 upper left corner and increase from left to
70. fail CAUTION FIRE HAZARD For continued protection against the risk of fire replace fuses only with listed and certified fuses of the same type and rating as those currently in the ViiA 7 Instrument Fuses 12 5A Time Lag T 250VAC 5 x 20 mm 2 Safety glasses Powder free gloves Screwdriver flathead Power off then unplug the ViiA 7 Instrument Allow it to cool for 15 minutes Using a flat head screwdriver unscrew and remove the fuse holder 8 Remove each fuse from its fuse holder and inspect it for damage Carbon typically coats the inside of failed fuses ra Ry Good Failed iil Replace each failed fuse with a 12 5A Time Lag T 250VAC 5 x 20 mm Fuse Note The voltage and amperage ratings are on the fuse holder Install the fuse holder Plug in then power on the ViiA 7 Instrument The installation is successful if the instrument powers on Note Fuse failure can result from fluctuations in the supplied power to the ViiA 7 Instrument To prevent further failures consider installing an electrical protective device such as a UPS or a surge protector Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter5 Service 5 Update the Windows operating system Update the Windows operating system Do not upgrade or update the Microsoft Windows operating system of the computer running the ViiA
71. for a sample with quantity equal to 1 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Numerics 128 ASCII character barcode support 26 7900 export file format 216 A accessories 176 account setup 104 suspended activate 107 suspension 104 122 user 106 action log contents 113 display 111 114 119 activation license keys 133 administrator auditing 110 password 101 security 101 user role 107 AIF 241 AIF 241 AIX 241 allele 241 altitude requirement 22 amplicon 241 amplification 241 amplification efficiency EFF 241 amplification plot 241 amplification stage 241 annual maintenance tasks 34 APIPA support 88 Applied Biosystems support 240 archive audit records 113 experiment files 37 instrument settings 37 148 arguments command line batch file creation 188 results exportation 189 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Index array card background creating 166 calibration 35 prepare for calibration 38 prepare for verification 78 Array Card RNase P Kit 75 177 assay 241 Assay ID 241 assay index file 241 assay information file 185 file format 198 assay information file AIF 242 audit administrators 110 action log 111 113 114 119 archive records 113 audit actions 113 audit mode 110 audit reason settings 110 audited objects and actions 110 enable or disabl
72. for individual wells and at individual cycles A solution that contains all components to run the PCR reaction except for the template sample standard or control Also called a PCR cocktail The PCR reaction components used to amplify the target and to detect amplification Process of collecting fluorescence data during PCR Data from the real time PCR are used to calculate results for quantification experiments or to troubleshoot results for genotyping or presence absence experiments A reporting format that is compliant with the Minimum Information for Publication for Quantitative Real Time Experiments MIQE guidelines In an HRM experiment the melt curve selected by a user in the difference plot to use as a basis for comparison The software displays the aligned data as the difference in fluorescence between the reference curve and the other melt curves In relative standard curve and Comparative Cr AAC7 experiments the sample used as the basis for relative quantification results Also called the calibrator The reference SNP refSNP cluster ID Generated by the Single Nucleotide Polymorphism Database of Nucleotide Sequence Variation dbSNP at the National Center for Biotechnology Information NCBI The refSNP ID can be used to search the Applied Biosystems Store for an Applied Biosystems SNP Genotyping Assay Also called an rs number Applied Biosystems ViiA 7 Real Time PCR System User Guide 247 Calibration Main
73. from assay information files aif which is shipped on a CD with each assay order The aif contains technical details about all assays in the shipment It includes information about assay concentrations reporters and quenchers used part and lot numbers and assay vial and plate ID numbers The file name includes the number from the bar code on the plate Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference Export formats and file specifications Export formats and file specifications This section describes the export formats supported by the ViiA 7 Software The information provided in this appendix is intended for users who want to integrate the ViiA 7 Software with third party applications including downstream analysis software and laboratory information management system LIMS tools Note The file specifications listed in this appendix are subject to change For updated information review the ViiA 7 Software Release Notes found at D AppliedBiosystems ViiA7 Software release notes html Export formats The ViiA 7 Software can export setup and results data from experiment files eds in several file formats that allow further downstream analysis The export formats feature standardized data structures and markup to maximize accessibility by downstream applications The ViiA 7 Software supports the following e
74. handle the 124 Perform this procedure to eliminate fluorescent contaminants from the ViiA 7 System sample block Contamination is generally evident in failed background calibrations where one or more wells consistently exhibit abnormally high signals CAUTION PHYSICAL INJURY HAZARD Do not remove the ViiA 7 Instrument cover There are no components inside the instrument that you can safely service yourself If you suspect a problem contact an Applied Biosystems Service Representative CAUTION PHYSICAL INJURY HAZARD During instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature CAUTION Before using a cleaning or decontamination method other than those recommended by the Applied Biosystems verify with Applied Biosystems that the proposed method will not damage the equipment e Bleach 10 solution e Tissue lint free e Cotton or nylon swabs and lint free cloths e Ethanol 95 solution e Safety glasses e Pipette 100 uL with pipette tips e Powder free gloves e Screwdriver e Deionized water sample block To prevent damaging or contaminating the sample block handle the assembly as shown Also when the assembly has been removed from the ViiA 7 Instrument place the sample block on a clean dry surface or in its shipping container Applied Biosystems ViiA 7 Real Time PCR System User
75. in the well UNKNOWN or NTC Reporter The reporter dye that labels the probe for the 100 character string target assay Quencher The quencher dye that labels the probe for the 100 character string target assay Rn The raw fluorescence for the associated well Float normalized to the fluorescence of the passive reference dye Rn Mean The averaged normalized fluorescence R for the Float associated replicate wells that contain the same target sample combination Rn SD The standard deviation of the normalized Float fluorescence R for the associated replicate wells that contain the same target sample combination Threshold The calculated value of the threshold for a positive Float Value call Call The presence absence call assigned tothe sample Negative Control at the specified well Blocked IPC Control IPC Failed Positive or Negative Comments Additional text that describes the well 1024 character string Automatic Ct Threshold Indicates whether the threshold was determined automatically true or manually false true Or false Ct Threshold The threshold cycle C for the sample at the well Float Automatic Ct Indicates whether the baseline was determined true Or false Baseline automatically true or manually false Baseline Start The first cycle used to calculate the baseline Float Baseline End The last cycle used to calculate the baseline Float Custom 1 Custom6 The
76. information from the Help system The ViiA 7 System has a Help system that describes how to use each feature of the user interface Access the Help system by doing one of the following e Click in the toolbar of the ViiA 7 Software window e Select Help gt ViiA 7 Software Help e Press F1 You can use the Help system to find topics of interest by e Reviewing the table of contents e Searching for a specific topic e Searching an alphabetized index Applied Biosystems ViiA 7 Real Time PCR System User Guide 239 Calibration Maintenance Networking and Security Documentation and Support Obtaining support Obtaining support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems website you can e Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Applied Biosystems user documents SDSs certificates of analysis and other related documents e Download PDF documents e Obtain information about customer training e Download software updates and patches 240 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security AIF AIX allele amplicon amplification amplification efficiency EFF amplification plot
77. instrument self test 000 0c ccc eee teenies 149 m Update the instrument firmware 1 6 66 eee eens 150 Note The touchscreen does not provide access to all instrument functions Features such as instrument calibration and remote notification are available only through the ViiA 7 Software Applied Biosystems ViiA 7 Real Time PCR System User Guide 147 Calibration Maintenance Networking and Security A Appendix A Manual Instrument Operation Maintain the instrument from the touchscreen Back up and restore the instrument settings Back up the ViiA 7 Instrument settings Restore the instrument settings 148 You can use the ViiA 7 Instrument touchscreen to back up the instrument settings icon standby time out and cover idle temperature and some network settings the Autodiscovery and Smart Monitoring options In the event that the ViiA 7 Instrument settings are reset you can restore the settings from the backup instrument settings from a USB Flash Drive Before backing up or restoring settings you must plug the drive into one of the USB ports behind the right side of the ViiA 7 Instrument touchscreen The ViiA 7 Instrument backs up to and restores ul IMPORTANT Do not use the USB ports on the rear panel of the ViiA 7 Instrument The rear USB ports are for use by Applied Biosystems personnel only to service the instrument y Note The backup feature c
78. is shipped on a CD with each TaqMan assay ordered from Applied Biosystems The file which contains data describing the assay can be imported into the ViiA 7 Software for use in related experiments page 198 Bar code file txt A user created text file containing the bar codes of consumables for which you want to create experiment files using the command line utility page 198 Conventions The following conventions are used in the rest of this section e normal Normal text must be entered exactly as it appears e lt italic gt Italicized text between brackets must be substituted with custom values e required text Text appearing between brackets is required information All information inside the brackets must be present for the ViiA 7 Software to import it e optional text Text appearing between braces is optional e Unless noted otherwise separate all fields in a row using a tab character U 0009 e Unless noted otherwise end all rows using a carriage return character U 000D Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 191 E Appendix E ViiA 7 Software Reference Import formats and file specifications Plate setup file format File structure Plate setup file header 192 You can use plate setup files to automatically populate setup information into an open experiment in the Vii
79. is stored in its original packaging sleeve you can use it to calibrate a ViiA 7 System 3 times for up to 6 months after you open it 9 Inthe Uniformity Calibration screen click Finish to complete the calibration then click Yes when prompted to save the results 58 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the uniformity calibration Troubleshoot the uniformity calibration Problem symptom Possible cause Action Uniformity calibration failed Abnormally low spectra across all wells of the plate or array card 1 Confirm that you loaded an RO plate or array card into the ViiA 7 Instrument If not perform the calibration again using the correct ROI plate or array card 2 If you are using the correct plate or array card perform the calibration again using a different ROI plate or array card 3 If the calibration fails again contact Applied Biosystems technical support One or more wells produced spectra that deviate significantly from the rest of the plate or array card 1 While viewing the calibration data in the Analysis screen locate the well s with abnormal signal in the Plate Layout tab 2 Rotate the calibration plate or array card 180 then perform the calibration again 3 Determine the location of the contaminated wells again If the position s of the wel
80. maximum capacity transfer the older data to another storage device Back up the instrument settings You can use the ViiA 7 Instrument touchscreen to back up the instrument settings instrument name icon standby time out and cover idle temperature In the event that the ViiA 7 Instrument settings are reset you can restore the settings from the backup See Back up the ViiA 7 Instrument settings on page 148 for more information Applied Biosystems ViiA 7 Real Time PCR System User Guide 37 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Fill the array cards Fill the array cards TM IMPORTANT Perform the following procedure only if you are calibrating a ViiA 7 System with an array card sample block Otherwise go to Perform the ROI calibration on page 42 to begin the calibrations Materials required Vii A 7 System Array Card Spectral Calibration Dye Kit Applied Biosystems Array Cards empty Array Card Spectral Calibration Dye Kit including FAM dye mix VIC dye mix ROX dye mix ROI dye mix Background Buffer FAM ROX dye mix and VIC ROX dye mix Applied Biosystems Array Card Staker Sealer Centrifuge with array card buckets and array card carrier clips Permanent marker or pen Pipettor 200 uL with pipette tips Powder free gloves Safety glasses Fill the calibration array cards IMPORTANT Wear powder free glo
81. necessary archive or back up your experiment files and instrument settings Power off the computer that controls the ViiA 7 System then after 30 seconds power on the computer Clean the surface of the ViiA 7 System with a lint free cloth Perform a ViiA 7 Instrument self test Monthly Check the lamp status If necessary replace the lamp Perform a background calibration t Run disk cleanup and disk defragmentation Semi annually Perform a regions of interest ROI calibration 6 months Perform a background calibration Perform a uniformity calibration Perform a dye calibration Perform a normalization calibration Perform an instrument verification run As needed Decontaminate the ViiA 7 System Replace the ViiA 7 System fuses Update the Windows operating system Update the ViiA 7 Software and firmware You can perform a background calibration to check for contamination If any parts of the optics are replaced or moved you must perform all calibrations including an RNase P instrument verification run 34 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security About the consumables Chapter 2 Calibration and Maintenance About the consumables The ViiA 7 System uses a series of specialized consumables for calibration The calibration plates and array cards can be ord
82. of primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target The nucleic acid sequence to amplify and detect In the software a color assigned to a target to identify the target in the plate layout and analysis plots In the software the type of reaction performed in the well for the target or SNP assay Available tasks e Unknown e Negative Control e Standard standard curve and relative standard curve experiments e Positive control genotyping experiments e IPC presence absence experiments e Blocked IPC presence absence experiments Wells containing identical reaction components including sample important for evaluating precision In the software a display of temperatures for the instrument cover and instrument block during the instrument run The type of nucleic acid to add to the PCR reaction A user created file that contains experiment setup information experiment type sample names target name and thermal conditions to be used as a starting point for new experiment setup Template files have an edt extension Part of the run method that specifies the temperature time ramp and data collection points for all steps and stages of the instrument run Applied Biosystems ViiA 7 Real Time PCR System User Guide 251 Calibration Maintenance Networking and Security Glossary threshold threshold cycle Cy touchscreen uniformity calibration u
83. only the outlier is flagged e Contamination e Improper sealing or seal leaks 1 Decontaminate the work area and pipettors 2 Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit Repeat the experiment and make sure to properly seal the RNase P plate or array card If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Well displays the SPIKE flag indicating the amplification curve contains one or more data points inconsistent with the other points in the curve e Bubbles in the reaction e Evaporation during the denaturation step because of improper sealing or seal leaks Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit Repeat the experiment and make sure to properly seal and centrifuge the RNase P plate or array card If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Well displays the THOLDFAIL flag indicating that the software cannot calculate the threshold e Amplification too early e Amplification too late e Low amplification e No amplification If a well is flagged confirm the results 1 Select the flagged well s in the plate layout or well table 2 View the amplification plot Rn vs Cycle and DRn vs Cycle and check for early late low or no amplification 3 Contact Applied Biosystems to order a replac
84. plate from its packaging Do not remove the optical film IMPORTANT Do not discard the packaging You can use the background plate to calibrate a ViiA 7 System 3 times for up to 6 months if it is stored in its original packaging sleeve 3 Vortex and centrifuge the background plate a Vortex the background plate for 5 seconds b Centrifuge the plate for 2 minutes at less than 1500 rpm IMPORTANT The background plate must be well mixed and centrifuged c Confirm that the liquid in each well of the background plate is at the bottom of the well If not centrifuge the plate again at a higher rpm and for a longer period of time IMPORTANT Do not allow the bottom of the plate to become dirty Fluids and other contaminants that adhere to the plate bottom can contaminate the sample block and cause an abnormally high background signal Correct Incorrect Liquid is at bottom of well e Not centrifuged with enough force or e Not centrifuged for enough time 50 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the background calibration Perform the calibration 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments Note You
85. plate layout select the wells containing Unknown Population A e 96 well plate Select rows A through C 5 000 copy population e 384 well plate Select columns 7 through 15 5 000 copy population e Array card Select rows C and D 800 copy population b In the plot verify that the Cys of the replicate population are equivalent Note The numbers on the X axis of the plot correspond to the wells of the reaction plate Beginning with well A1 the wells are numbered from left to right and top to bottom Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Verify the instrument performance c If an outlier is present in the selected population select the corresponding well of the plate layout then click Omit to remove the well from the analysis If the total number of outliers for the replicate population exceeds the limit in the table below repeat the experiment using another RNase P plate or array card Maximum number of outlier wells that can be removed Sample block Unknown Unknown Standard No template population Af population B STD controls NTC 96 well plate 6 6 1 0 384 well plate 10 10 2 0 Array card 4 4 4 0 5 000 copy population for 96 384 well plates 800 copy population for array cards 10 000 copy population for 96 384 well plates 1 600 copy population for array cards
86. powder free gloves and safety glasses when you prepare plates or array cards IMPORTANT Before performing a normalization calibration you must perform ROL background uniformity and dye calibrations Prepare the calibration consumables appropriate for your ViiA 7 Instrument Prepare the normalization plates 0 0 cece eee eee see below Fill the array Cards iiccesin vs pia kenurna ueiaire odin apaaw asd dhaad aad 4 38 Remove the normalization plates from the freezer then allow the plates to warm to room temperature approximately 5 minutes IMPORTANT Do not remove the normalization plates from their packaging until you are ready to run them The fluorescent dyes in the dye plates are photosensitive Prolonged exposure to light can diminish the fluorescence signal strength of the plates Note If you store the normalization plates in their original packaging and in the freezer you can use them to calibrate a ViiA 7 System up to 3 times for 6 months after opening them Go to Perform the calibration on page 71 Before using each normalization plate vortex the plate for 5 seconds centrifuge it for 2 minutes at less than 1500 rpm then verify that the liquid in each dye plate is at the bottom of the wells If not centrifuge the plate again at a higher rpm and for a longer period of time Correct Incorrect Liquid is at e Not centrifuged with enough force or bottom of well e Not
87. repeat the calibration using a different background plate If the calibration fails again determine the source of the contamination as explained in How to identify contamination on page 54 Instrument does not eject the background plate The adhesive cover may have adhered the plate to the heated cover within the instrument If the plate does not eject power off Wearing powder free gloves reach Perform a background calibration to Power off the ViiA 7 Instrument Wait for 15 minutes then power on the ViiA 7 Instrument and eject the plate and unplug the ViiA 7 Instrument then open the access door into the ViiA 7 Instrument and remove the plate from the heated cover then close the access door confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a local Applied Biosystems Field Service Office Applied Biosystems ViiA 7 Real Time PCR System User Guide 53 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the background calibration How to identify contamination 54 Signals that exceed the limit of normal background fluorescence may indicate fluorescent contaminants on the calibration plate or the sample block Common contaminants include ink residue from permanent pens powder from disposable gloves and dust To determine the so
88. replicate wells for the Float specified target sample combination Ct SD The standard deviation of the average Cy of the Float replicate wells for the specified target Automatic Ct Indicates whether the baseline was determined TRUE Or FALSE Baseline automatically true or manually false Baseline Start The first cycle used to calculate the baseline Integer Baseline End The last cycle used to calculate the baseline Integer Automatic Ct Indicates whether the threshold was determined TRUE Or FALSE Threshold automatically true or manually false Ct Threshold The threshold cycle C7 for the sample at the well Float Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom 220 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference Export formats and file specifications Genotyping experiments The following table describes the results data exported from genotyping experiments Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Sample Name The name of the sample contained by the well 100 character string SNP Assay The name of the SNP assay added to the well 100 character string Name Allele1 Rn The raw fluorescence associated with the allele 1 Float probe of the SNP assay at
89. right and from top to bottom Contains red b lue and g reen color values each between 0 to 255 The field is enclosed in double quotes with no spaces between the values Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference Export formats and file specifications Genotyping experiments The table below describes the sample setup data that can be exported from a genotyping experiment The body can contain all or some of the data columns below depending on the export configuration Note For all other experiments see Quantification and presence absence experiments on page 202 Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 t Sample Name The name of the sample contained by the well 100 character string Sample Color The RGB color of the associated sample RGB lt r gt lt g gt lt b gt 8 SNP Assay The name of the SNP assay applied to the well 100 character string Name If the well contains multiple assays the data for each SNP assay are exported in an additional row SNP Assay The RGB color of the associated SNP assay RGB lt r gt lt g gt lt b gt Color Task The task assignment of the SNP assay at the UNKNOWN Or NTC well Allele1 Name The name of the first allele for the associated SNP assay
90. sample names from the SampleFileNames txt sample file Generates an experiment for each bar code in the bar codes v12 txt bar code file where each new experiment uses the settings found in the standard_curve edt template file and the spS_820629 txt setup file Note The setup file links the information from the AIF_820629 txt and SampleFileNames txt to each new experiment file Saves all generated files using the following naming convention lt barcode gt _alloptionsused Saves all generated files to C ViiA7 Experiment lt date time gt Note The command line application automatically creates a time stamped folder at the export location for each batch operation For example the folder created for files generated on April 7 2010 at 12 48 35 would be 2010 04 07 124835 The following example performs a real time data markup language RDML export of experiments in the ViiA 7 Software experiments directory to the exports directory of the C drive The software generates an RDML file for each individual experiment file cmdlineutil exe export e D Applied Biosystems ViiA7 Software vi l experniments e SDS23 1 C exports s single a rdmil Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference E Import formats and file specifications Import formats and file specifications The ViiA 7
91. that are repeated Fluorescence data collected during the extension stage are displayed in an amplification plot and the data are used to calculate results With TaqMan chemistry the last two steps of a PCR stage are typically combined See also cycling stage assay In a PCR reaction mix two target specific primers or two primers and a probe used to amplify a target Assay ID Identifier assigned by Applied Biosystems to TaqMan assays Applied Biosystems ViiA 7 Real Time PCR System User Guide 241 Calibration Maintenance Networking and Security Glossary assay information file AIF background calibration baseline baseline corrected normalized reporter ARn baseline threshold algorithm biological replicates blocked IPC calibrator chemistry Cr 242 Tab delimited data file on a CD shipped with each assay order The AIF contains technical details about all assays in the shipment It includes information about assay concentrations reporters and quenchers used part and lot numbers and assay vial and plate ID numbers The file name includes the number from the bar code on the plate Type of calibration in which the instrument performs reads of a background plate averages the spectra recorded during the run and extracts the resulting spectral component to a calibration file The software then uses the calibration file during subsequent runs to remove the background fluorescence from the run data
92. the ViiA 7 Instrument then touch Done The instrument name is the alphanumeric string used to identify the ViiA 7 Instrument on the network IMPORTANT To connect the ViiA 7 Instrument to a network the name must be unique IMPORTANT The instrument name cannot include spaces or special characters such as lt gt 1 Upload the instrument icon The instrument icon is the graphic used to represent the ViiA 7 Instrument in the ViiA 7 Software Instrument Console a Save the replacement graphic to a USB drive then plug the drive into the USB port on the right side of the ViiA 7 Instrument touchscreen b Touch Upload Icon select the desired graphic file then touch Done Note The replacement graphic must be a maximum of 48 x 48 pixels and be stored in the portable net graphic PNG format Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security C Appendix A Manual Instrument Operation A Administrate the instrument from the touchscreen Unplug the USB drive Define the standby time out setting a b C Select Standby Time out to activate the feature Touch the Standby Time out field Enter the number of minutes 1 to 300 that the ViiA 7 Instrument should remain idle until it enters standby mode then touch Done TM Note When in standby mode the ViiA 7 Instrument powers off the LCD scree
93. the two targets CCKAR and GH1 targets are added to the same well For both TaqMan assays the probes are labeled with the FAM reporter dye and the non fluorescent quencher NFQ MGB Instrument Type ViiA 7 Passive Reference ROX Sample Setup Well Sample Name Sample Color Biogroup Name Biogroup Color Target Name Target Color Task Reporter Quencher Quantity Comments le Liver cDNA RGB 257 0 10 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB aL Liver cDNA TRGB 25 070 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 2 Liver cDNA RGB 257070 er CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 2 Liver cDNA RGB 25 0 0 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB a Liver cDNA TRGB 25 070 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 3 Liver cDNA RGB 257 070 S GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 4 Heart cDNA RGB 0 25 0 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 4 Heart cDNA RGB 0 25 0 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 5 Heart cDNA RGB 0 25 0 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 5 Heart cDNA RGB 0 25 0 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 6 Heart cDNA RGB 0 25 0 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 6 Heart cDNA RGB 0 2570 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB i Brain cDNA RGB 0 0 25 7 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB T Brain cDNA RGB 0 0 25 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 8 Brain cDNA RGB 0 0 25 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 8 Brain cDNA RGB
94. the PCR 4 Load the appropriate dye plate into the plate adapter select Please check the box when the dye calibration plate has been loaded click Next twice then click START RUN to start the calibration 5 When the run is complete and the ViiA 7 Instrument ejects the plate remove and discard the plate or array card WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can be heated to 100 C Before removing the plate be sure to wait until it reaches room temperature 6 Inthe Dye dialog box of the ViiA 7 Software click Next Applied Biosystems ViiA 7 Real Time PCR System User Guide 171 Calibration Maintenance Networking and Security 172 Appendix C Creating Custom Calibration Plates and Array Cards Create a custom dye plate for calibration 7 Verify the grouping of the dye spectra a In the plate layout select the wells of the plate b Inspect the raw data For each spectrum verify that the peak is e Within the detectable range for the ViiA 7 System e Free of irregular spectral peaks e Present at the correct filter for the dye Note Among wells containing the same dye variations in spectral position and peak position are caused by minor differences in the optical properties and excitation energy between the individual wells Verify the status of the calibration If the calibration Passed If all spectra are acceptable finish the calibration a Click Next b Enter
95. the well normalized to the fluorescence of the passive reference dye Allele2 Rn The raw fluorescence associated with the allele 2 Float probe of the SNP assay at the well normalized to the fluorescence of the passive reference dye Call The allele call assigned to the sample at the Homozygous specified well lt allele x allele x gt Heterozygous lt allele x allele y gt or Negative Control NC Quality The confidence of the automatic allele call Float 1 to 100 Method The method used to call alleles Auto Or Manual Task The task assigned to the target in the well UNKNOWN or NTC Pass Ref The raw fluorescence of passive reference at the Float well Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 221 E Appendix E ViiA 7 Software Reference Export formats and file specifications RDML export format For more information 222 The ViiA 7 Software can export data from real time quantitative PCR experiments as well formed Real time PCR Data Markup Language RDML a structured extensible markup language XML standard for quantitative PCR qPCR data In combination with the Minimal Information MIQPCR guidelines the RDML element structure describes all aspects of a qPCR experiment including setup analysis and data interpretation
96. them to warm to room temperature approximately 5 minutes IMPORTANT Do not remove the dye plates from their packaging until you are ready to run them The dyes in the dye plates are photosensitive Prolonged exposure to light can diminish the fluorescence signal strength of the plates Note If you store Applied Biosystems dye plates in their original packaging and in the freezer you can use them to calibrate a ViiA 7 System up to 3 times for 6 months after opening them Before using each dye plate vortex the plate for 5 seconds centrifuge it for 2 minutes at less than 1500 rpm then confirm that the liquid in each dye plate is at the bottom of the wells If not centrifuge the plate again at a higher rpm and for a longer period of time Correct Incorrect a Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time IMPORTANT The dye plates must be well mixed and centrifuged Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the dye calibration Perform the calibration IMPORTANT The ViiA 7 Software guides you through the calibration of each dye separately You must set up run and analyze each dye independently 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instr
97. to tab delimited text files txt in a legacy export format of the Applied Biosystems 7900HT Real Time PCR System The 7900 export format features a standardized data structure and markup to maximize accessibility by downstream applications Data exported in the ViiA 7 export format can be opened by common spreadsheet applications such as Microsoft Excel or imported by laboratory information management system LIMS applications that have been configured to parse the file format TM Note Due to the very different nature of the ViiA 7 Instrument some export types are not available Note Column customization sorting and omission is not available Only multiple tab delimited text files are supported The following table shows the data files that the ViiA 7 Software can export in the 7900 export format Each row represents a single exportable data file File Description See Setup file Describes the configuration of samples on the experiment page 217 consumable including sample location target or SNP assay properties and task assignments Multicomponent Contains the spectral data used by the ViiA 7 Software to page 218 file generate the multicomponent plot that displays the contribution of each dye over the duration of the PCR run Results file Contains the normalized processed and analyzed data page 219 generated by the ViiA 7 Software Applied Biosystems ViiA 7 Real Time PCR System U
98. with the ViiA 7 System computer and for network communication When the ViiA 7 System is connected to a network computers on the network that run the ViiA 7 Software can e Send and download experiments to from the ViiA 7 System e Run experiments on the ViiA 7 System e Remote monitor the ViiA 7 System as it performs runs The Ethernet 1 port of the ViiA 7 Instrument supports e Static IP network service with subnet mask primary and secondary data network service DNS and default gateway settings or dynamic host configuration protocol DHCP network service e mDNS DNS for local domains Note Because mDNS is limited to direct network connections a ViiA 7 System set for mDNS may not be visible to other nodes that are separated by a router hub or another network device e IPv4 link local IPV4LL in the RFC also known as Automatic Private IP Addressing APIPA or Internet Protocol Automatic Configuration IPAC Note When the ViiA 7 System is set for DHCP APIPA is automatically enabled and the ViiA 7 System provides an IP address when no address is supplied by the DHCP server Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 3 Networking 3 Networking overview Example network layouts Example 1 Example 2 In the following example a one or more ViiA 7 Instruments which have been configured for dynamic host co
99. 000 cece eect ete eee eeee 229 Workstation safety sieaas 204 omean eee Ses od OR Gees dbo be AE eased 229 Safety and electromagnetic compatibility EMC standards 230 Chemical safety 22200 coed heat ead Sele ee a bee ie St iene 231 General chemical safety 0 0 c cece eect eens 231 SDS Skia ct aie cc cahat aN e Acai de han eda Aglare bel Sultan 2 ith ith ute ty ees 232 Chemical waste safety 0 0 cece cece eee eee eee nett eeeees 232 Biological hazard safety 0 c cece cece eect eee eee 234 Safety alent Soia 2s heen ha cee A eee atlas et Maire ee ele detec eg 235 General alerts forall chemicalS 0 0 0 eee eee cee aranana 235 General alerts for instrumentation 0c cece eee e eee eee eee eee 235 Specific alerts for instrumentation 2 0000 cece eee eee eens 235 Documentation and Support 0e cece e eee eee 237 Related documentation ecsedi sarasa eese eee eee eee eee eee eee eens 237 Obtaining information from the Help system 000c cece eee eee ees 239 Obtaining SUPPO T 24 30 secs ed a ee eee r ten ets AE 240 Glossary oe eee ea ee ate eee ail esate nina ae 241 Index sien a cet ee ee poeta deece red dere 253 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security About This Guide Purpose The Applied Biosystems ViiA 7 Real Time PCR System User Guide provides refere
100. 100 character string Allele1 Color The RGB color of the first allele for the associated SNP assay RGB lt r gt lt g gt lt b gt Allele1 The reporter dye that labels the probe for the 100 character string Reporter first allele Allele1 The quencher dye that labels the probe for the 100 character string Quencher first allele Allele2 Name The name of the second allele for the associated SNP assay 100 character string Allele2 Color The RGB color of the second allele for the associated SNP assay RGB lt r gt lt g gt lt b gt 8 Allele2 The reporter dye that labels the probe for the 100 character string Reporter second allele Allele2 The quencher dye that labels the probe for the 100 character string Quencher second allele Comments Additional text that describes the well 1024 character string Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Contains rJed blue and g reen color values each between 0 to 255 The field is enclosed in double quotes with no spaces between the values Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 203 E Appendix E ViiA 7 Software Reference Export formats and file specifications Raw data The ViiA 7 Software can export the unprocessed raw data R collected by the ViiA 7 Instrument during t
101. 2 Quencher The quencher dye used to label the probe for the second allele lt dye name gt 88 t See the Applied Biosystems ViiA 7 Real Time PCR System Getting Started Guide to determine the tasks applicable to your experiment Cannot be blank Contains rJed b lue and g reen color values between 0 to 255 The field must be set within double quotes with no spaces between the values ttCan be empty if the Task field is empty Otherwise the field must contain a value The dye must already exist in the ViiA 7 Software Dye Library The dye name must be 100 characters or less 194 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security AyluNnIas pue bulysomjany aueuajulep uoljesgHeg Faping J SN WAS S YOd awil jeay Z VIIA swiajsksoig panddy G l Examples Quantitative PCR experiments The following example shows a plate setup file created for a quantitative PCR experiment to be run on a ViiA 7 System The experiment evaluates the expression of two targets CCKAR and GH1 in three samples CDNA from the liver heart and brain For both TaqMar assays the probes are labeled with the FAM reporter dye and the non fluorescent quencher NFQ MGB Biological groups are not used in this experiment Instrument Type ViiA 7 Passive Reference ROX Sample Setup Well Sample Name Sample Color Biogroup Name Biogroup Color Target Name
102. 20 notification 104 105 overview 101 security policies 104 spaces in user names 104 user accounts 106 user name restrictions 104 user report 109 user role 107 security auditing and electronic signature module See audit See security security users account suspension 122 login 121 overview 121 password change 121 permissions 121 session timeout 122 self test performing 149 serial factor 249 serial port 25 28 service pack updates 131 session timeout 104 122 set up instrument security 156 software security 103 settings date time 152 instrument name 152 instrument security 156 maintenance reminders 153 network instrument 154 system shortcuts 155 setup file 185 signing electronic signature 122 slope 249 Smart Monitoring 154 SMTP requirement 96 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security software instrument 30 licenses maintenance 133 third party 31 software update 132 spatial calibration signing 122 specification halogen lamp 127 installation 76 specifications installation 76 instrument 20 stage 249 staker sealer 38 181 standard 249 standard curve 250 standard curve method 250 standard quantity 250 standards EMC 230 safety 230 standby mode 160 standby time out 152 starting quantity 250 static IP support 88 statistics instrument 155 status lamp 128 step 250 storage instrument 162 surge protector requirements 29 SYBR Green
103. 3 24 PORT A Con 1 D A 800 2 E own B44 600 3 e Standard 4 J Sta 5 Sta l 6 M tand 7 p Sta 3 8 For each transfer do the following a Place the array card on a lab bench with the foil side down b Load 100 uL of fluid into a pipette c Hold the pipette in an angled position 45 degrees and place the tip into the fill port There is a fill port on the left arm of each fill reservoir the larger of the two holes IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port 78 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance 2 Verify the instrument performance d Dispense the fluid so that it sweeps in and around the fill reservoir toward the vent port When pipetting the reagents into the array card pipette the entire 100 uL volume into the fill reservoir but do not go past the first stop of pipettor plunger or you may blow the solution out of the port IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port 5 Centrifuge and seal the array card as explained in steps 6 through 11 on page 40 6 Run the prepared array card as soon as possible after filling it Store the array card in a dark place until you are ready to run it IMPORTANT Do not expose the array card to light until you are ready to run
104. 5 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the dye calibration 7 After loading the plate or array card start the calibration a In the Dye Calibration screen select Check the box when the dye calibration plate has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the ViiA 7 Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature 8 When the run is complete and the ViiA 7 Software displays the Analysis screen confirm the grouping of the dye spectra a Select the Plate Layout tab then review the raw data For each spectrum verify that the peak is e Within the detectable range for the ViiA 7 System e Free of irregular spectral peaks e Present at the correct filter for the dye see the following table Filter set x1 m1 x2 m2 x3 m3 x4 m4 x5 m5 x6 m6 Blue Green Yellow Orange Red Deep red Excitation nm 470415 520 10 549 5410 580 10 640 10 662 10 Emission nm 520 15 558 12 586 5 10 623 14 682 14 711 12 System dyes FAM dye HEX dye e NED dye ROX dye LIZ dye SYBR JOE dye e TAMRA Green dye TET dye dye VIC dye 66 Note Among wells containing the same dye varia
105. 7 Software without first consulting the software release notes or the Applied Biosystems website Future versions and updates to the Windows operating system can conflict with the ViiA 7 Software To determine compatibility of an upgrade or update 1 Open D Applied Biosystems ViiA7 Software double click release notes html then read the ViiA 7 Software Release Notes for the compatibility of interest 2 Ifthe release notes do not mention the compatibility use an internet browser to visit info appliedbiosystems com ViiA7 then search the website for the compatibility of interest 3 If the website does not contain the information of interest contact Applied Biosystems Applied Biosystems ViiA 7 Real Time PCR System User Guide 131 Calibration Maintenance Networking and Security 5 Chapter 5 Service Update the ViiA 7 Software and Firmware Update the ViiA 7 Software and Firmware Applied Biosystems may release updates to the ViiA 7 Software and ViiA 7 Instrument firmware that you can install without the aid of Applied Biosystems service personnel You can obtain updates directly from the service section of the Applied Biosystems website For the latest services and support information for the ViiA 7 System 1 Go to https www 2 appliedbiosystems com support software 2 In the Software Downloads page select Applied Biosystems ViiA 7 Real Time PCR System from the menu 3 In the ViiA 7 Instrume
106. 7 System performs the tests successfully perform experiments normally If the ViiA 7 System encounters errors during the test runs the software may not be compatible with the ViiA 7 Software Applied Biosystems ViiA 7 Real Time PCR System User Guide 31 Calibration Maintenance Networking and Security Chapter 1 Getting Started Using this guide Using this guide 32 You can use this guide to calibrate service network and administrate the Applied Biosystems ViiA 7 Real Time PCR System This user guide contains the following information Chapter 2 Calibration and Maintenance Describes how to perform regular maintenance of the ViiA 7 System including calibrating the ViiA 7 Instrument and verifying instrument performance Chapter 3 Networking Describes how to install the ViiA 7 System to a local area network for remote monitoring and control Chapter 4 Security Audit and Electronic Signature Describes how to configure the security audit and e signature functions of the ViiA 7 Software Chapter 5 Service Describes how to replace the user serviceable parts of the ViiA 7 Instrument and resolve infrequent problems that can occur during normal use Appendix A Manual Instrument Operation Describes how to operate the ViiA 7 Instrument manually using the touchscreen interface Appendix B Power On or Off Store and Move the ViiA 7 System
107. 73 Howto orde skate Oh Sc siti ct a oA ce Te ens othe Se nome tee Ra oe ote Nelda 174 How to order from the ViiA 7 Software 0 0 0 ccc cece cece cence ene ens 174 How to order from the Applied Biosystems Website 22220000055 175 ACCESSOMES agate eae dane a Ree aw wait a eae ra a ale Bad a deste 176 Calibration and verification kits nnna ccc ccc cence ee cece nee n ee eees 177 384 well sample block kits 2 2 2 2 0c 177 96 well sample block kits 0 0 20 cece cece ett eens 178 Fast 96 well sample block kits nnna ananunua ccc cece ences 179 Array card sample block kits 2 0 0 0 c cece eee eee eens 180 Consumables s sinerien taaa bebe Slee ae Med nh led ot del ee dee 181 APPENDIX E ViiA 7 Software Reference cccecceeeeeeeeeeees 183 ViiA 7 Software command line application 2 0 00 c ccc ccc cence ences 184 Command line workflowS 000s ccc cence cee eee n ene enn enes 184 Supporting files for experiment creation 00 00 cece eee ee eee eens 185 Precedence rules for experiment file generation 2000000000 eee 186 Running the command line application from a command prompt 187 Command syntax and arguments n aesan nannan cece eee e eee eee eeeeee 188 Examples in un guest cece Seine tye Garlen Wee A e ae I eee he ae teed 190 Import formats and file specifications 00 cee eee eee aes 191 About the import file for
108. A 7 Software or into new experiments created by the command line application see page 184 A plate setup file is a tab delimited ASCII text file txt that contains data that describes the location experiment data information The files can be created manually using a text processor or generated automatically by third party applications IMPORTANT To guarantee successful import of the plate setup file into a experiment the file must contain all the elements described in the following section and in the order that they appear The plate setup file consists of a header which specifies the instrument model for which the experiment is designed and a sample setup section Section Description See Plate setup file header Defines the instrument model for which the page 192 experiment is designed and the dye used as the passive reference Plate setup file body Defines the contents of a 96 384 well plate or array page 193 card including target SNP assay sample and task assignments The plate setup file begins with a header that consists of two lines Each line starts with an asterisk and ends with a carriage return in the following pattern lt field name gt lt field value gt The header must contain the lines shown in the following table Field Description Valid Values Instrument The model of ViiA 7 System for ViiA 7 Type which the experiment is designed Passive The dye that th
109. A 7 System Table 1 Applied Biosystems ViiA 7 Real Time PCR System Documentation Document Purpose and audience Applied Biosystems ViiA 7 Real Time PCR System Site Preparation Guide PN 4445302 Explains how to prepare your site to receive and install the ViiA 7 System Intended for personnel who schedule manage and perform the tasks required to prepare your site for installation of the ViiA 7 System Applied Biosystems ViiA 7 Real Time PCR System Quick Reference Card PN 4448987 Explains how to perform genotyping and gene expression experiments using the ViiA 7 System Intended for laboratory staff who perform experiments using the ViiA 7 System Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security PN 4442661 Explains how to calibrate maintain network and secure the ViiA 7 System Intended for laboratory staff who maintain the ViiA 7 System Applied Biosystems ViiA 7 Real Time PCR System Getting Started Guide PN 4441434 Explains how to perform experiments on the ViiA 7 System The guide functions as both a e Tutorial using example experiment data provided with the ViiA 7 System e Guide for your own experiments Intended for laboratory staff who perform experiments using the ViiA 7 System Applied Biosystems ViiA 7 Real Time PCR System Automation Accessory Guide
110. ANT You must perform a background calibration before every series of dye calibrations Because the age and use of instrument components can affect spectra readings we recommend performing a dye calibration at least every 6 months Applied Biosystems ViiA 7 Real Time PCR System User Guide 61 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Perform the dye calibration About the dye calibration System dyes Custom dyes 62 The Applied Biosystems ViiA 7 Real Time PCR System calibrates the following system dyes FAM dye NED dye ROX dye SYBR Green dye TAMRA dye and VIC dye The following figure shows the emission spectrum for each dye and the filters and wavelengths at which each dye is read Fitters eee l 500 600 700 Wavelength nm Emission Spectra Dyes 520 nm 550 nm 580 nm 610 nm FAM VIC NED ROX SYBR Green TAMRA The ViiA 7 System can be used to run assays designed with custom dyes dyes not supplied by Applied Biosystems however before using custom dyes with the ViiA 7 System you must create and run a custom calibration plate The ViiA 7 Software uses the custom calibration plate to create a spectral standard to distinguish the custom dye in the fluorescence data collected during the run See Create a custom dye plate for calibration on page 168 for information on custom dye calibrations IMPORTANT A custom dye mu
111. Audit and Electronic Signature changes and actions Note You can enable or disable auditing globally and by record type It is disabled globally by default e Electronic signature e sig Determines if users are required to provide a user name and password when performing certain functions You can configure e sig so that a user can print a report or start a run only if the associated data are signed You can also configure each e sig event to require multiple signatures and to require users with specific permissions to sign Note Electronic signature can be enabled or disabled globally It is disabled globally by default Applied Biosystems ViiA 7 Real Time PCR System User Guide 101 Calibration Maintenance Networking and Security 4 Chapter 4 Security Audit and Electronic Signature Administrators overview Example applications You can configure the SAE module in a variety of ways For example you can e Require users to log in and leave audit disabled e Allow only certain users to create or modify protocols e Allow only certain users to approve reviewed samples e Require experiments to be signed before users can run or print them 102 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators Configure the security system Configure the security system Access the Security screen and enable or disable security Use the Security scree
112. BR Green dyes x2 m2 Green 520 10 558 12 VIC JOE TET and HEX dyes x3 m3 Yellow 549 5410 586 5410 NED and TAMRA dyes x4 m4 Orange 580 10 623 14 ROX dye x5 m5 Red 640 10 682414 LIZ dye x6 m6 Deep red 662 10 711412 None amp t The central wavelengths are the optimized wavelengths No Applied Biosystems supported dye currently available Custom dyes The Applied Biosystems ViiA 7 Real Time PCR System can run assays designed with custom dyes dyes not supplied by Applied Biosystems that are excited between 455 to 672 nm and read between 505 to 723 nm Applied Biosystems ViiA 7 Real Time PCR System User Guide 19 Calibration Maintenance Networking and Security 1 Chapter 1 Getting Started Specifications and layout Specifications and layout ViiA 7 System specifications The figures below summarize the specifications and requirements for the ViiA 7 System For more information refer to the Applied Biosystems ViiA 7 Real Time PCR System Site Preparation Guide PN 4445302 BOR ty eee ert a Sih ade iat BB en oe ede oe Ech ate ge hal A antares icin ViA 7 System 15 24 cm 6 in dimensions i o i oH oO 1 Yo 33 g as iN a a o Be 2 a 53 3 cm 21i 162 56 cm 64 in a Q 3 8 A 5 Width Depth Height Weight Component cm in cm in cm in kg lbs Instrument 53 3 21 0 63 5 25 0
113. GB 2 Pos Control RGB 0 25 0 E coli RGB 98 2570 1 TEC FAM NFQ MGB 5 Pos Control RGB 0 25 0 IPC RGB 98 25 0 IPC VIC NFQ MGB 6 Pos Control RGB 0 25 0 E coli RGB 98 25 0 IPC FAM NFQ MGB 6 Pos Control RGB 0 25 0 TEG RGB 989 2570 2 TPC MEC NFQ MGB 7 Blocked IPC RGB 0 0 25 E coli RGB 98 25 0 BlockedIPC FAM NFQ MGB a Blocked IPC RGB 0 0 25 TPC RGB 0 07 L05 1 BliockedIPC VIC NFQ MGB 8 Blocked IPC RGB 0 0 25 E coli RGB 0 0 105 BlockedIPC FAM NFQ MGB 8 Blocked IPC RGB 0 0 25 TEC RGB 0 0 105 BlockedIPC VIC NFQ MGB 9 Blocked IPC RGB 0 0 25 E coli RGB 0 0 105 BlockedIPC FAM NFQ MGB 9 Blocked IPC RGB 0 0 25 IPC RGB 0 0 105 BlockedIPC VIC NFQ MGB 10 Sample01 RGB 90 0 0 E coli RGB 0 0 105 UNKNOWN FAM NFQ MGB 10 Sample01 RGB 90 0 0 IPC RGB 0 0 105 UNKNOWN Wane NFQ MGB ir Sample01 SERGE O01 OO ie E coli RGB 0 0 105 UNKNOWN FAM NFQ MGB Ski Sample01 RGB 90 0 0 IPC RGB 0 0 105 UNKNOWN vic NFQ MGB i Sample01 RGB 90 0 0 E coli RGB 0 0 105 UNKNOWN FAM NFQ MGB 12 Sample01 RGB 90 0 0 IPC RGB 0 0 105 UNKNOWN VIC NFQ MGB Genotyping experiments The following example shows a plate setup file created for a genotyping experiment to be run on a ViiA 7 System The experiment screens samples for one SNP targets rs15934 using a set of allele specific TaqMan probes labeled with the FAM and VIC reporter dyes and the non fluorescent quencher NFQ MGB Instrument Type
114. Guide Calibration Maintenance Networking and Security Chapter5 Service 5 Decontaminate the sample block Clean the sample block WARNING PHYSICAL INJURY HAZARD During instrument operation the sample block and heated cover can be heated to 100 C Before removing the sample block be sure to wait until it reaches room temperature IMPORTANT Wear powder free gloves when you perform this procedure 1 Identify the contaminated wells of the sample block see How to identify contamination on page 54 2 Power off and unplug the ViiA 7 Instrument then allow it to cool for 15 minutes 3 Open the access door 4 Firmly press down on the handle of the sample block then remove it from the ViiA 7 Instrument Place the sample block on a clean dry surface 5 Clean the contaminated wells of the sample block using deionized water a Pipette a small volume of deionized water into each contaminated well b In each well pipette the water up and down several times to rinse the well c Pipette the water to a waste beaker d Using a cotton swab scrub inside of each contaminated well e Using a lint free cloth absorb the excess deionized water Applied Biosystems ViiA 7 Real Time PCR System User Guide 125 Calibration Maintenance Networking and Security 126 Chapter5 Service Decontaminate the sample block 10 11 12 13 14 Load the sample block into the ViiA 7 Instr
115. HAZARD Severe electrical shock can result from operating the ViiA 7 Instrument without its instrument panels in place Do not remove instrument panels High voltage contacts are exposed when instrument panels are removed from the instrument WARNING FIRE HAZARD Improper fuses or high voltage supply can damage the instrument wiring system and cause a fire Before turning on the instrument verify that the fuses are properly installed and that the instrument voltage matches the power supply in your laboratory WARNING FIRE HAZARD For continued protection against the risk of fire replace fuses only with fuses of the type and rating specified for the instrument WARNING ELECTRICAL HAZARD Grounding circuit continuity is required for the safe operation of equipment Never operate equipment with the grounding conductor disconnected WARNING ELECTRICAL HAZARD Use properly configured and approved line cords for the voltage supply in your facility WARNING ELECTRICAL HAZARD Plug the system into a properly grounded receptacle with adequate current capacity The ViiA 7 System has an installation overvoltage category of II and is classified as portable equipment Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix F Safety F Instrumentation safety Bar code scanner laser safety Laser The bar code scanners included with the ViiA 7 Ins
116. Log In and Submit Order Materials Log In f Log into the Applied Biosystems Store to place the selected items in your shopping basket If you do not have a user name and password click Register Now to create a new account Store Log In OR Register To log into the Applied Biosystems Store enter your user name Ifyou do not and password then click Log In and Submit have an Applied Biosystems User Name account click create a new Password account Log In and Submit the link below ta Register Now I Remember my user name and password for future orders Note If you do not have an account with the Applied Biosystems Online Store click Register Now to create an account When you are connected to the Applied Biosystems Store follow the prompts to complete your order How to order from the Applied Biosystems Website To order Procedure Assays and reagents 1 Go to www appliedbiosystems com 2 Under I Want to Buy select the product of interest Instrument parts and accessories Calibration kits 1 2 3 Go to info appliedbiosystems com ViiA7 Click Parts and accessories Select the desired components the complete the order as instructed See Consumables on page 181 for a complete list of compatible instrument parts accessories and kits Applied Biosystems ViiA 7 Real Time PCR System User Guide 175 Calibration Mai
117. N 4 1 rh 1 Section 4 1 Administrators Administrators overview IMPORTANT The Security Audit and Electronic Signature SAE module is installed only with Applied Biosystems ViiA 7 Real Time PCR Systems that were purchased with the SAE module IMPORTANT Enabling the Security Audit and Electronic Signature module alone does not make the ViiA 7 System compliant with any particular standard You must modify the module settings according to your requirements to ensure compliance The Security Audit and Electronic Signature SAE module is an optional component of the ViiA 7 Software that can allow you to configure the ViiA 7 System to meet specific requirements The module provides the following functionality e Security Controls user access to the software A default Administrator user account is provided and additional user accounts and permissions can be user defined Note The default password for the Administrator user account is Administrator however the password can be changed during installation Note You can enable or disable system security globally e Auditing Tracks changes made to library items actions performed by users and changes to the Security Audit and Electronic Signature settings The software automatically audits some actions silently You can select other items for auditing and specify the audit mode The Auditing function provides reports for audited library items Security
118. Networking and Security Chapter 2 Calibration and Maintenance Perform the background calibration 7 When the run is complete and the ViiA 7 Software displays the Analysis screen confirm the analysis status of the calibration then select the QC tab and review the quality check summary e Analysis Status Indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or that the data it did collect is unusable Note Abnormal spectra or abnormally high background fluorescence can indicate the presence of contamination on the plate array card or sample block which can cause the calibration to fail e QC Status Indicates the quality of the calibration data where passed indicates that all wells produced data that passed the quality check and failed indicates that one or more wells produced spectra that deviate significantly from the other wells on the plate Analysis ac Action status status Passed Passed Click Next then remove the plate or array card when the ViiA 7 Instrument ejects the tray arm Passed Failed Troubleshoot the failed calibration as described in Troubleshoot the background calibration on page 53 Failed Failed Note You can accept a calibration that passes the Analysis Status check but fails the QC Status check We recommend using calibrations that yield passing results for both s
119. Open the Calibration Reminders screen for the ViiA 7 Instrument a In the Home screen of the ViiA 7 Software click Instrument Console b In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a ViiA 7 Instrument to your list before you can manage it c After the ViiA 7 Instrument is added to your list select it then click Manage Instrument d In the Instrument Manager click Maintenance then click Calibration Reminders 2 In the Calibration Reminders Setting table configure the notification settings for the calibrations in interest For each calibration that you want to monitor a In the Expiry Interval column enter the number of days that elapse before the type of calibration expires on the ViiA 7 Instrument b In the Send a Reminder column select the check box to configure the ViiA 7 Software to email a reminder to perform the calibration c In the Reminder Interval column enter the number of days that elapse before the software emails recipients a reminder to perform the calibration 3 In the Enter e mail addresses for notifications field enter the email address es that you want to receive email notifications Separate multiple email addresses with commas 4 Click Apply to change the notification settings Applied Biosystems ViiA 7 Real Time PCR System User Guide 97 Calibration Mai
120. PC gt a Reporter The reporter dye used by the associated target assay lt dye name gt tt88 x Quencher The quencher dye used by the associated target assay lt dye name gt 88 lt Quantity Optional The quantity of standard present in the given well lt float or Integer gt expressed as a float or integer If the associated well is not assigned the STANDARD task then the field is blank SNP Assay Name The name of the SNP assay detected or amplified by the assay lt 100 character string gt t in the associated well SNP Assay Color Optional SNP assay color in RGB RGB lt r gt lt g gt lt b gt Task The task assignment of the SNP assay at the well lt UNKNOWN NTC PC_ALLELE_1 PC_ALLELE_2 gt PC_ALLELE_BOTH gt 5 Allele1 Name The name of the first allele detected by the SNP assay lt 100 character string gt tt oD Allele1 Color The RGB color used to represent data for the first allele RGB lt r gt lt g gt lt b gt u t gt Allele Reporter The reporter dye used to label the probe for the first allele lt dye name gt tt88 v lt dye name gt 88 Allele2 Name The name of the second allele detected by the SNP assay lt 100 character string gt Allele2 Color The RGB color used to represent data for the second allele RGB lt r gt lt g gt lt b gt Allele2 Reporter The reporter dye used to label the probe for the second allele lt dye name gt tt88 Allele
121. ROI calibration plate for 5 seconds b Centrifuge the plate for 2 minutes at less than 1500 rpm IMPORTANT The ROI calibration plate must be well mixed and centrifuged c Verify that the liquid in each well of the ROI calibration plate is at the bottom of the well If not centrifuge the plate again at a higher rpm and for a longer period of time Correct Incorrect Liquid is at e Not centrifuged with enough force or bottom of well Not centrifuged for enough time 44 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the RO calibration Perform the calibration 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a ViiA 7 Instrument to your list before you can manage it 3 After the ViiA 7 Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenance then click ROI b In the ROI Calibration screen click Start Calibration 5 Click Next then perform the calibration as instructed When the side door opens load the ROI calibration plate or array card Ensure that the plate or array card is properly aligned in the
122. RTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 6 After loading the plate or array card start the calibration a In the Setup tab select Check the box when the Uniformity Calibration plate has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the ViiA 7 Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature Applied Biosystems ViiA 7 Real Time PCR System User Guide 57 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Perform the uniformity calibration 7 When the run is complete and the ViiA 7 Software displays the Analysis screen confirm the analysis status of the calibration then select the QC tab and review the quality check summary e Analysis Status Indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or that the data it did collect is unusable Note A calibration can fail if wells produce spectra that deviate significantly from the other wells of the plate or if all wells produce abnormally low spectra Abnormal spectra can indicate the presence of fl
123. Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 1 Getting Started 1 About the ViiA 7 System 2 Emission The ViiA 7 Instrument optics collect the residual fluorescence emitted from the wells of the reaction plate The resulting image collected by the device consists only of light that corresponds to the range of emission wavelengths 3 Collection The ViiA 7 Instrument assembles a digital representation of the residual fluorescence collected over a fixed time interval The ViiA 7 Software stores the raw fluorescent image for analysis After a run the ViiA 7 Software uses calibration data ROI background uniformity dye and normalization to determine the location and intensity of the fluorescent signals in each read the dye associated with each fluorescent signal and the significance of the signal Instrument filters and supported dyes System dyes The Applied Biosystems ViiA 7 Real Time PCR System features a six color filter set that supports all Applied Biosystems dyes The following figure shows the emission spectrum for each dye and the filter at which each dye is read Fitters TC 500 600 700 Wavelength nm Emission Spectra Dyes 520 nm 550 nm 580 nm 610 nm FAM VIC NED ROX SYBR Green TAMRA Filter Filter wavelength nm set Color Supported dyes Excitation Emission x1 m1 Blue 470 15 520415 FAM and SY
124. Sample file format on page 197 for more information Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 185 E Appendix E ViiA 7 Software Reference ViiA 7 Software command line application Precedence rules for experiment file generation When generating experiment files eds the ViiA 7 Software command line interface relies on a set of precedence rules to resolve conflicts that arise from the data supplied by some input files Assay information files aif or aix plate setup files txt and template files edt can contain data used to populate the same fields of new experiment files For example both template and plate setup files can contain location data for samples and assays Files used for experiment file eds creation Precedence rule Template file edt The values in the template take precedence except for e Experiment Name Determined by the File Name Convention preference e Bar Code Determined by the bar code if present Otherwise the value is null e Experiment File Name Determined by the File Name Convention preference e Template file edt e Assay information file aif aix All values in the template file take precedence except for e Gene Expression Targets Assay Definition e Genotyping Assay SNP Definition e Passive Reference If any conflicts exist between the assay information file and the temp
125. Security Chapter 2 Calibration and Maintenance Verify the instrument performance Prepare the TagMan RNase P plate or array card IMPORTANT When performing the RNase P instrument verification experiment e Perform all calibrations beforehand e Run the TaqMan RNase P plate or array card soon after you allow the plate or reagents to thaw Minimizing the time that the prepared consumable sits on the bench ensures optimal performance e Wear powder free gloves and safety glasses when you prepare plates or array cards Prepare the instrument verification consumable appropriate for your instrument m Prepare the TaqMan RNase P Instrument Verification Plate see below m Prepare the array card for the instrument verification run 78 Pre pare the 1 Obtain the TaqMan RNase P Instrument Verification Plate from the freezer then Taq Man RNase P allow the plate to warm to room temperature for approximately 5 minutes Instrument Verification Plate IMPORTANT Do not remove the plate from its packaging until you are ready to run it The fluorescent dyes in the dye plate are photosensitive Prolonged exposure to light can diminish the fluorescence signal strength of the plate 2 Remove the RNase P plate from its packaging 3 Briefly vortex and centrifuge the RNase P plate a Vortex the plate for 5 seconds b Centrifuge the reaction plate for 2 minutes at less than 1500 rpm IMPORTANT The reactio
126. Software supports several import file formats that can be used to automate experiment creation and assay and sample data import The files can be used with the command line application see page 184 or the ViiA 7 Software application programming interface API to integrate the ViiA 7 System into a laboratory information management system LIMS For a detailed explanation of the API or for information on integrating the ViiA 7 System into a laboratory workflow see the Applied Biosystems ViiA 7 Real Time PCR System Robotics User Guide PN 4442663 Note The file specifications listed in this appendix are subject to change For updated information review the ViiA 7 Software Release Notes found at D AppliedBiosystems ViiA7 Software release notes html About the import file formats File format Description See Plate setup file txt A user created tab delimited text file that describes the layout of a consumable for an experiment to be run on the ViiA 7 System The file defines the arrangement of assays and samples on the consumable and provides other experiment data such as the thermal profile and data collection settings page 192 Sample file txt A user created tab delimited text file containing sample data that can be imported into the ViiA 7 Software for use in related experiments page 197 Assay information file aif or aix A tab delimited or XML data file that
127. System or select Start gt All Programs gt Applied Biosystems gt ViiA 7 System gt ViiA 7 Software d If the ViiA 7 Software Login appears enter your user name and password then click OK 160 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix B Power On or Off Store and Move the ViiA 7 System Power off the ViiA 7 System Power off the ViiA 7 System The Applied Biosystems ViiA 7 Real Time PCR System operates in low power mode when not in use however the ViiA 7 System can be powered off completely so that the components draw no power Note If the ViiA 7 System will be inactive for extended period of time prepare it for storage as explained in Store the ViiA 7 System on page 162 To power off the ViiA 7 System components 1 Power off the ViiA 7 Instrument a If the ViiA 7 Instrument touchscreen is not blank touch f8 to place the ViiA 7 Instrument into stand by mode b Toggle the power button on the rear of the ViiA 7 Instrument 2 Power off the ViiA 7 System computer a In the desktop select Start Shut Down b In the Shut Down Windows dialog box select Shut Down then click OK 3 Power off the monitor 4 If you have an Applied Biosystems Twister II Robot toggle the power button on the rear of the Twister II Robot Applied Biosystems ViiA 7 Real Time PCR System User Gui
128. Target Color Task Reporter Quencher Quantity Comments al Liver cDNA RGB 25 0 0 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 2 Liver cDNA RGB 25 0 0 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 3 Liver cDNA RGB 297 0r 0 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 4 Heart cDNA TRGEI0 25 0 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 3 Heart cDNA RGB 0 25 0 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 6 Heart cDNA RGB 0 25 10 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 7 Brain cDNA RGB 0 0 25 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 8 Brain cDNA RGB 070 25 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 9 Brain cDNA RGB 0 0 25 CCKAR RGB 98 25 0 ENDOGENOUS FAM NFQ MGB 376 Liver cDNA TRGB 25 0 10 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB Sieh Liver cDNA RGB 25 0 0 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 378 Liver cDNA RGB 25 0 0 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 379 Heart cDNA TRGBTO 257 0 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 380 Heart cDNA RGB 0 25 0 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 381 Heart cDNA RGB 0 25 0 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 382 Brain cDNA RGB 07 0 25 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 383 Brain cDNA RGB 0 0 25 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB 384 Brain cDNA RGB 0 025 GH1 RGB 0 0 105 UNKNOWN FAM NFQ MGB The following example shows a plate setup file for a multiplex version of the experiment above where the assays for
129. The exported RDML data is saved as a flat text file that can be used to transfer qPCR data between the ViiA 7 Software and third party applications IMPORTANT The RDML export format is available only for standard curve gene expression and relative standard curve experiments The RDML standard is maintained by the RDML consortium an organization that consists of key developer groups and a member community For more information on the RDML format visit the RDM organization website www rdml org The website features free data management tools including an on line RDML file generator and RDML software libraries Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security APPENDIX F Safety This appendix covers E Instrumentation safety 0 eens 224 Symbols on instruments prisa sne npe ate EES eee nee EE 224 Locations of safety labels on instruments 0 0c eee eee eee eee 226 General instrument safety 000 e eee eee eens 227 Physical hazard safety 2 vis os 9805 mesia Shee Teds BE es eae 228 Electrical safety es ccs aided doria e iiaia alee ig audible ai a maiaa lad does 228 Bar code scanner laser safety 1 0 0 6 6c cece nee eens 229 Workstation safety semer pirana tti ee dliio EENES RETENEN E easy os 229 Safety and electromagnetic compatibility EMC standards 230 M Chemical Safety reires reiron Eae eens 231 General che
130. User Guide Calibration Maintenance Networking and Security 87 Chapter 3 Networking Networking overview Networking overview After installing the ViiA 7 System you can connect the ViiA 7 System to a local area network to enhance its functionality This chapter describes how to e Set up the ViiA 7 System for use on a network e Set up a computer for remote monitoring e Test the network connection by engaging the remote monitoring feature Controlling and monitoring networked ViiA 7 Instruments When the ViiA 7 Instrument is connected to a network computers on the network that are running the ViiA 7 Software can control or monitor it The ViiA 7 Software can control up to 4 instruments and monitor up to 15 instruments simultaneously A networked ViiA 7 Instrument can be controlled by only one computer at a time A networked computer running the ViiA 7 Software can transfer experiments to and from an instrument begin or stop a run and perform some maintenance functions During a run the Remote Monitoring feature of the software can be used to view the run status temperature and amplification data in real time See Monitor the ViiA 7 Instrument on page 94 for more information on remote monitoring Note Remote monitoring does not allow you to control the ViiA 7 System About the Ethernet 1 port 88 The ViiA 7 Instrument features a Gigabit Ethernet 1 port for direct communication
131. a with noticeably high precision among technical replicates An illustration of the grid of wells and assigned content in the reaction plate The number of rows and columns in the grid depends on the sample block that you use In the software you can use the plate layout as a selection tool to assign well contents to view well assignments and to view results The plate layout can be printed included in a report exported and saved as a slide for a presentation A file txt csv xml or sds that contains setup information such as the well number sample name sample color target name dyes and other reaction plate contents One standard in a standard curve The standard quantity for each point in a standard curve is calculated based on the starting quantity and serial factor In genotyping and presence absence experiments a DNA sample with a known genotype homozygous or heterozygous In the software the task for the SNP assay in wells that contain a sample with a known genotype PCR reaction component that contains the forward primer and reverse primer designed to amplify the target PCR reaction component that contains the primers designed to amplify the target and a TaqMar probe designed to detect amplification of the target Fluorescent compound used to calibrate the instrument See system dye Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security quanti
132. administration functions are for systems administrators or for information technology personnel The right most column indicates whether a function is available when the ViiA 7 Instrument is operating in secure mode see Enable or disable instrument security on page 156 for more information Available in User role Function secure mode Operational Create experiments from templates No Run experiments Transfer experiments templates and results to from a USB drive Maintenance Back up and restore the instrument settings Yes Perform an instrument self test Update the ViiA 7 Instrument firmware Administration Define the date and time Define the instrument settings Define the network settings Define the maintenance reminders Define the system shortcuts Enable or disable instrument security Review the instrument statistics View the ViiA 7 Instrument log Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix A Manual Instrument Operation A Operate the instrument from the touchscreen Operate the instrument from the touchscreen The touchscreen provides limited control of the ViiA 7 Instrument to run experiments and transfer data You can perform the following functions from the touchscreen to operate the ViiA 7 Instrument without using the ViiA 7 Software m Create an exp
133. age copy number of unknown population B OCopyUnk2 Standard deviation of unknown population B Note Unknown population A refers to the 5 000 copy population in columns 7 through 15 of the TaqMan RNase P Plate or the 800 copy population in rows C and D of the loaded array card Unknown population B refers to the 10 000 copy population in the wells of the TaqMan RNase P Plate or the 1 600 copy population in rows E and F of the loaded array card Installation specification The ViiA 7 System passes the installation specification if the inequality holds and the ViiA 7 Instrument successfully distinguishes between unknown populations A and B with a statistical confidence level of 99 7 As shown in the following table you can omit a limited number of outlier wells from the unknown populations to meet the installation specification Maximum number of outlier wells that can be removed Sample block Unknown Unknown Standards No template population At population B STD controls NTC 96 well plate 6 6 1 0 384 well plate 10 10 2 0 Array card 4 4 4 0 5 000 copy population for 384 well plates 800 copy population for array cards 10 000 copy population for 384 well plates 1 400 copy population for array cards Maximum number of wells that can be removed from each standard population 76 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and
134. al optional and last name Click a field to display the field limits which are specified in Security settings Note First name MI middle initial and last name are used to create User Full Name which is displayed as the name of the logged in user Note You cannot change the user name after you save the user account Select Password Expires at First Login to require the user account to specify a new password at first log in The Password Expires On date is specified in Security settings Select the user role described in Create or edit a user role on page 107 and the electronic signature state determines if a user account has permission to electronically sign objects Leave the status set to ACTIVE Optional Enter email for information only phone and comments Click Save A grayed Save button indicates an invalid entry in a field Click a field to display the limits for the field then enter a valid entry Inthe Users screen select a user account then click Edit Note If you select multiple users only Status and Role will be changed Edit settings as needed You cannot edit the user name of an existing user Click Save Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators 4 Manage user accounts Activate a 1 In the Users screen select the user suspended user account 2 Click Edit 3
135. alibration and Maintenance Perform the normalization calibration Troubleshoot the normalization calibration Problem symptom Possible cause Action Normalization calibration failed Abnormally low spectra across all wells of the plate or array card 1 Confirm that you loaded an normalization plate or array card into the ViiA 7 Instrument If not perform the calibration again using the correct normalization plate or array card 2 If you are using the correct plate or array card perform the calibration again using a different normalization plate or array card 3 If the calibration fails again contact Applied Biosystems technical support One or more wells produced spectra that deviate significantly from the rest of the plate or array card 1 While viewing the calibration data locate the well s with abnormal signal in the Plate Layout tab 2 Rotate the calibration plate or array card 180 then perform the calibration again 3 Determine the location of the contaminated wells again If the position s of the well s identified in steps 1 and 2 are e Identical The sample block is contaminated Decontaminate the sample block see Decontaminate the sample block on page 124 e Reversed The normalization plate or array card is contaminated Discard the plate or array card then perform the normalization calibration using a new normalization plate or array card 4 If the calibration fa
136. an be used as an administrative tool to manage ViiA 7 Instruments You can use the feature to create a standard image for a ViiA 7 Instrument that can then be restored on other instruments to bypass the manual setup process 1 Plug a USB drive into the USB port on the right side of the ViiA 7 Instrument touchscreen 2 If the instrument is in standby touch the ViiA 7 Instrument touchscreen to awaken it then touch 3 In the Main Menu touch x Tools then touch Back Up Settings 4 In the Backup Settings screen touch Y Backup 5 Touch to return to the Main Menu 6 Unplug the USB drive Note For administrative purposes you can reuse the instrument settings saved to the USB drive to configure more than one ViiA 7 Instruments Note that you must configure the network settings for each instrument individually 1 Plug the USB drive that contains the instrument settings into the USB port on the right side of the ViiA 7 Instrument touchscreen 2 Ifthe instrument is in standby touch the ViiA 7 Instrument touchscreen to awaken it then press Ot 3 In the Main Menu touch gt lt Tools then touch Restore Settings Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix A Manual Instrument Operation A Maintain the instrument from the touchscreen 4 In the Restore Settings screen select the settings to restore a Touch the setting
137. analysis results audit records and other plate specific data Experiment documents have the suffixes eds experiment document single edt template and edm multiple Applied Biosystems ViiA 7 Real Time PCR System User Guide 243 Calibration Maintenance Networking and Security Glossary experiment name Experiment Setup experiment type export filter flag forward primer holding stage housekeeping gene import 244 Entered during experiment setup the name that is used to identify the experiment A software feature that allows you to set up an experiment according to your experiment design Experiment Setup provides you with maximum flexibility in the design and setup of your experiment The type of experiment to perform e Standard curve e Comparative Cy AACT e Relative standard curve e Genotyping e Presence absence e Melt curve The experiment type that you select affects setup run and analysis A software feature that allows you to export experiment setup files experiment results instrument information and security and auditing settings to spreadsheet presentation or text files You can edit the default location of the exported file Dye excitation and emission filter combination that you select for an experiment The ViiA 7 System includes a six color filter set that supports FAM NED ROX SYBR Green TAMRA and VIC dyes A quality control QC indicator w
138. applied A Bee iene by Life technologies Applied Biosystems VIIA 7 Real Time PCR System Calibration Maintenance Networking and Security User Guide gt applied h Biosystems by Life technologies Applied Biosystems VIIA 7 Real Time PCR System Calibration Maintenance Networking and Security User Guide For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES NOTICE TO PURCHASER Label License No right is conveyed expressly by implication or by estoppel under any other patent claim such as claims to apparatus reagents kits or methods such as 5 nuclease methods other than apparatus claims covering the purchased apparatus Further information on purchasing licenses may be obtai
139. art Run or Print Report The e signature system is configured to require signatures from two users from the user account named Administrator before a user can start a run The experiment has not been signed A user attempts to begin the run The following message is displayed Missing Required Signatures dd The requested operation cannot be performed because some required signatures are missing The Following e Signatures must be collected before this operation can be performed Before the run can start two administrators must sign If a user with an incorrect user role signs the message is displayed again Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators Generate e signature reports Generate e signature reports You can use the ViiA 7 Software to generate reports of e signature history from open experiment eds or template edt files Display the e sig records 1 In the ViiA 7 Software open an experiment eds or template edt file ZA 2 In the open experiment or template click S Audit then click E Signatures 3 Optional Click any row to view details for individual signatures Save or print e sig records 1 In the ViiA 7 Software open an experiment eds or template edt file 2 In the open experiment or template click o Audit then click E Signatures 3 In the table select the record to be saved
140. as WINS define the settings a Click Advanced in the Internet Protocol TCP IP Properties dialog box b Define the IP Settings DNS and WINS tabs as instructed by your systems administrator then click OK 8 Close all dialog boxes by clicking OK 9 Restart the computer The computer is now visible to other computers on the network Install the ViiA 7 Software 1 If you are using a computer that you have provided install the ViiA 7 Software using the Applied Biosystems ViiA 7 Software CD Note You must install the ViiA 7 Software to monitor the ViiA 7 System over the network 2 Optional Install protective software to the computer Applied Biosystems ViiA 7 Real Time PCR System User Guide 93 Calibration Maintenance Networking and Security 3 Chapter 3 Networking Monitor the ViiA 7 Instrument Monitor the ViiA 7 Instrument After connecting the ViiA 7 System and a computer to the network you can enable remote monitoring in the ViiA 7 Software to observe the instrument status remotely About remote monitoring When the ViiA 7 System is connected to the network any computer on the network that is running the ViiA 7 Software can m Monitor the status of ViiA 7 Instrument during arun see below m Upload or download an experiment or template to a ViiA 7 Instrument 95 m Enable or change the calibration reminders 0 c ccc eee 96 Guidelines fo
141. as determined true Or false Baseline automatically true or manually false Baseline Start The first cycle used to calculate the baseline Integer Baseline End The last cycle used to calculate the baseline Integer Tm1 Tm3 The first second and third melting temperatures Float Tm calculated in degrees Celsius Comments Additional text that describes the well 1024 character string Custom The contents of the custom text fields foundinthe 1024 characterstring Custom Results table of the experiment per field Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 211 Export formats and file specifications Appendix E ViiA 7 Software Reference Presence absence The following table describes the results data exported from presence absence experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Sample Name The name of the sample contained by the well 100 character string Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target
142. ate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the bar code facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 7 After loading the plate or array card start the calibration a In the Dye Calibration screen select Check the box when the normalization calibration plate has been loaded then click Next b In the Run screen click START RUN to start the calibration IMPORTANT Do not attempt to open the access door during the run The door is locked while the ViiA 7 Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature Applied Biosystems ViiA 7 Real Time PCR System User Guide 71 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Perform the normalization calibration 8 When the run is complete and the ViiA 7 Software displays the Analysis screen verify the status of the calibration The analysis status indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or that the data it did
143. below IMPORTANT An internet connection a web browser and a valid email account are required to activate a ViiA 7 Software license If the computer that contains the ViiA 7 Software is not connected to the internet or it lacks an web browser application contact Applied Biosystems support to request the license file 1 In the main menu of the ViiA 7 Software select Tools License Central 2 In the License Central dialog box select the license of interest from the table click Renew License then wait for the default web browser application to connect to the Applied Biosystems website 3 In the Applied Biosystems Software License Activation website click ViiA 7 Software from the list of products then activate the license as instructed After you successfully activate the license the Applied Biosystems website emails you the activated license file lic to install on the computer After you activate your license and receive an activated license file lic install the file as explained below to unlock the ViiA 7 Software or module Note Each license file is generated specifically for the computer that was used to activate the license key 1 Save the license lic file to the computer that contains the ViiA 7 Software 2 In the main menu of the ViiA 7 Software select Tools License Central 3 In the License Central dialog box click Install License 4 In the Open dialog box navigate to and sele
144. ber of target assays on the consumable formatted as follows xxx Number of Detectors lt number of assays gt The column header defines the columns of exported data followed by one or more lines where each row defines the properties of a single assay separated by tab characters Column Description Output Detector The name of one target in the well if applicable 100 character string If a well contains multiple targets one row is used per target Reporter The reporter dye that labels the probe for the 100 character string target assay Quencher The quencher dye that labels the probe for the 100 character string target assay Description The standard 1024 character string Comments The additional text that describes the well 1024 character string Applied Biosystems ViiA 7 Real Time PCR System User Guide 217 Calibration Maintenance Networking and Security Multicomponent file 218 Appendix E ViiA 7 Software Reference Export formats and file specifications Well data After the assay data the ViiA 7 Software exports the well data that describes the configuration of samples and assays on the experiment consumable The table below describes the well data that can be exported from absolute quantification relative quantification or presence absence experiments If a well contains more than one assay the ViiA 7 Software lists the setup data for each additional assay in additional columns to the right of the existing data
145. bration 000 c ccc eee ees 69 m Verify the instrument performance 6 66 c cece eens 74 Access the Help system by pressing F1 by clicking in the toolbar of the ViiA 7 Software window or by selecting Help gt Contents and Index Applied Biosystems ViiA 7 Real Time PCR System User Guide 33 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Calibrate and maintain the ViiA 7 System Calibrate and maintain the ViiA 7 System The Applied Biosystems ViiA 7 Real Time PCR System requires regular calibration and maintenance for proper operation This chapter contains the procedures that you must perform on a regular basis to ensure optimal instrument performance Recommended calibration and maintenance schedule The following table displays the recommended maintenance schedule for ViiA 7 System users To ensure proper operation of your ViiA 7 Instrument perform the regular weekly monthly and semiannual maintenance indicated below IMPORTANT Calibrate the ViiA 7 System at the same ambient temperature at which you will run experiments Extreme variations in ambient temperature can affect the heating and cooling of the ViiA 7 System and in extreme cases influence experimental results IMPORTANT Do not use organic solvents to clean the ViiA 7 System Frequency User performed maintenance task Weekly Check the computer disk space If
146. card Prepare the ROI calibration plate 56 IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or array cards If you have an ROI plate or array card from a recent ROI calibration go to step 3b on page 56 plates or go to Perform the calibration on page 57 array cards Otherwise prepare the ROI calibration consumable appropriate for your ViiA 7 Instrument m Prepare the ROI calibration plate 0 eee see below E Fillithe array Cards icc a sce tiaia is aia a ka MEAE ee Aa EEEE 38 1 Remove the ROI calibration plate from the freezer then allow it to warm to room temperature approximately 5 minutes IMPORTANT Do not remove a calibration plate from its packaging until you are ready to run it The fluorescent dyes in the wells of the plate are photosensitive Prolonged exposure to light can diminish the fluorescence of the dye Remove the ROI calibration plate from its packaging Do not remove the optical film IMPORTANT Do not discard the packaging for the calibration plate You can use the plate to calibrate a ViiA 7 System 3 times for up to 6 months if it is stored in its sleeve Vortex and centrifuge the plate a Vortex the ROI calibration plate for 5 seconds b Centrifuge the plate for 2 minutes at less than 1500 rpm IMPORTANT The ROI calibration plate must be well mixed and centrifuged c Confirm that the liquid in each well of the ROI calibration plat
147. centrifuged for enough time IMPORTANT The normalization plates must be well mixed and centrifuged Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the normalization calibration Perform the calibration 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a ViiA 7 Instrument to your list before you can manage it 3 After the ViiA 7 Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenance then click Normalization b In the Normalization Calibration screen click Start Calibration 5 In the Normalization Calibration screen select the reporter passive dye combination that you want to calibrate then perform the calibration as instructed 6 Load the calibration plate or array card into the ViiA 7 Instrument a Verify that the normalization plate or array card matches the selection in the ViiA 7 Software The name of the dyes contained by each consumable appears next to the bar code on the front of the plate or array card b Load the appropriate normalization plate or array card into the plate adapter Ensure that the pl
148. cessite lutilisation d outils instrument ne contient aucune pi ce r parable par Uutilisateur Toute intervention doit tre effectu e par le personnel de service qualifi venant de chez Applied Biosystems CAUTION Moving parts Crush pinch hazard ATTENTION Pi ces en mouvement risque de pincement et ou d crasement Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 15 About This Guide Safety information 16 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security CHAPTER 1 Getting Started This chapter covers m About the ViIIA 7 System 6 net eens 18 E Specifications and layout 6 66 eee eens 20 mM ViiA 7 System hardware 6 eee nnn 24 E ViiA 7 System software sesa 0 eee aE 30 M Using this guide 0 ene ete e i 32 Access the Help system by pressing F1 by clicking in the toolbar of the ViiA 7 Software window or by selecting Help gt Contents and Index Applied Biosystems ViiA 7 Real Time PCR System User Guide 17 Calibration Maintenance Networking and Security 1 Chapter 1 Getting Started About the ViiA 7 System About the ViiA 7 System The Applied Biosystems ViiA 7 Real Time PCR System uses fluorescent based polymerase chain reaction PCR reagents to provide e Quantitative research detection of targe
149. collect is unusable Analysis status Action Passed Enter any comments you have in the Comments field click Next then remove the plate or array card when the ViiA 7 Instrument ejects the tray arm Failed Troubleshoot the failed calibration as described in Troubleshoot the normalization calibration on page 73 WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can be heated to 100 C Before removing the plate wait until it reaches room temperature IMPORTANT If the ViiA 7 Instrument does not eject the plate remove the plate as explained in Troubleshoot the normalization calibration on page 73 9 Discard or store the plate or array card Consumable Action Array card Discard the array card Plate Return the normalization calibration plate to its packaging sleeve then return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate is stored in its original packaging sleeve you can use it to calibrate a ViiA 7 System 3 times for up to 6 months after you open it 10 In the Normalization Calibration screen click Finish to complete the calibration then click Yes when prompted to save the results 11 Repeat steps 4 through 10 to perform the remaining normalization calibration 72 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 C
150. count activate suspended 107 create or edit 106 delete 107 inactivate 107 permissions 107 user role create 107 V verification array cards 78 consumables 181 kits 177 plate 77 VIC dye 19 62 ViiA 7 export file format 200 W WARNING description 14 warnings lamp 127 waste disposal guidelines 233 waste profiles description 233 weekly maintenance tasks 34 weight instrument 20 workstation safety 229 Y y intercept 252 262 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Part Number 4442661 Rev C 12 2010 444266190 gt applied A biosystems by Lefe technologies Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 760 603 7200 www lifetechnologies com Technical Resources and Support For the latest technical resources and support information for all locations please refer to our Web site at www appliedbiosystems com support
151. count for the specified period of inactivity 4 Select the notification method Notify Admin at Login If an event triggers notification the next time any user with an Administrator role logs in the software lists those events indicating the time each event occurred and the user who triggered the event The Administrator has the option of acknowledging the event which removes it from the notification list Email Notification If an event triggers notification the ViiA 7 Software sends an email to the addresses in the adjoining Email Address column of the table The email notification displays the triggered event and displays the time that the event occurred and the user who triggered the event 5 Click OK Applied Biosystems ViiA 7 Real Time PCR System User Guide 105 Calibration Maintenance Networking and Security 4 Chapter 4 Security Audit and Electronic Signature Manage user accounts Manage user accounts Create or edit a user account Create a user account Edit a user account 106 The software includes a default Administrator user account with permissions defined by the account user role to perform all functions in the software You cannot modify this account Inthe ViiA 7 Software select Tools gt Security gt Settings In the Security Settings dialog box select the Users tab Click Create to display the New User dialog box Enter user name password first name middle initi
152. creen select the update package then touch Upgrade Firmware Allow the ViiA 7 Instrument to complete the upgrade IMPORTANT Do not remove the USB drive from the ViiA 7 Instrument until you are instructed to do so 6 After the upgrade is complete and the ViiA 7 Instrument reboots confirm the upgrade success a Unplug the USB drive b Touch Settings then touch About this instrument to view the software version number to confirm that the firmware has been upgraded 150 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix A Manual Instrument Operation Administrate the instrument from the touchscreen Administrate the instrument from the touchscreen The touchscreen provides access to several administrative functions that you can use to integrate the ViiA 7 Instrument into a laboratory workflow The following functions are available from the touchscreen and can be used after installation to customize the ViiA 7 Instrument settings and configure it for network use E Define the date and time 6 66 eee eens Define the instrument settings 06 cece eee eens Define the maintenance reminders 0 006 cece ete eee Define the network settings 06 Define the system shortcuts 66 c cece neces Review the instrument statistics 0 0 0 cece cee cee eee eens Enable or disable instrument security s
153. ct the folder that contains the files that you want to download b In the Experiments field select the files that you want to download To select multiple files Ctrl click or Shift click files in the list c When you have selected the files that you want to download click Download d In the Send experiment to instrument dialog box select the folder to which you want to download the selected file s then click Open Note You can also use the Folders and Experiments fields to e Create or remove directories on the ViiA 7 Instrument e Add delete or download experiments on the ViiA 7 Instrument Applied Biosystems ViiA 7 Real Time PCR System User Guide 95 Calibration Maintenance Networking and Security 3 Chapter 3 Networking Monitor the ViiA 7 Instrument Enable or change the calibration reminders The calibration reminders settings allow you to configure the ViiA 7 Software to alert you by email when the ViiA 7 Instrument requires calibration The notifications settings feature is optional and it does not affect performance IMPORTANT The ViiA 7 Software transmits email only while the ViiA 7 Instrument is monitored If the network connection is interrupted the software stops transmitting updates Collect the required The ViiA 7 Software requires access to a Simple Mail Transfer Protocol SMTP server information to email calibration reminders Contact your systems administrator or informati
154. ct the license file then click Open 5 Click OK to close the License Central dialog box Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter5 Service 5 Replace the sample block Replace the sample block Replace the sample block in the event of a hardware failure or to change the consumable format of the ViiA 7 Instrument WARNING PHYSICAL INJURY HAZARD During instrument operation the sample block and heated cover can be heated to 100 C Before removing the sample block be sure to wait until it reaches room temperature Materials required e Safety glasses e Powder free gloves e Sample block How to handle the sample block To prevent damaging or contaminating the sample block handle the assembly as shown below After the assembly has been removed from the ViiA 7 Instrument place the sample block on a clean dry surface or in its shipping container Replace the sample block IMPORTANT If you are installing a sample block of a different format for example 96 384 well plate to array card you must also change the plate adapter to match the new consumable format 1 Power off and unplug the ViiA 7 Instrument then allow it to cool for 15 minutes 2 Open the access door Applied Biosystems ViiA 7 Real Time PCR System User Guide 135 Calibration Maintenance Networking and Security 136 Chapter 5 Service Replace
155. dd the custom dye to the software 1 Start the dye calibration a In the Home screen of the ViiA 7 Software click Instrument Console b In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments c Select the ViiA 7 Instrument then click Manage Instrument d In the Instrument Manager click Maintenance then click Dye e In the Background Calibration screen click Start Calibration 2 In the Dye window select a custom dye from the list or create the custom dye a Click New Dye b In the Dye Library dialog box click New c Complete the New Dye dialog box then click OK Field option Action Name Enter a name for the custom dye Wavelength Enter the wavelength at which the dye fluoresces Type Select e Reporter if the dye works in conjunction with a quencher dye to report an increase of PCR product e Quencher if the dye suppresses the fluorescence of a reporter dye until amplification of PCR product e Both if the dye reports an increase of PCR product without the aid of a quencher dye d Click Close 3 In the Dye window enter a temperature setting for the calibration Set the temperature to match the temperature at which you intend to collect data For example the temperature for all Applied Biosystems system dyes is 60 C because data collection for TaqMan reagents occurs during the 60 C extension step of
156. de 161 Calibration Maintenance Networking and Security B Appendix B Power On or Off Store and Move the ViiA 7 System Store the ViiA 7 System Store the ViiA 7 System The Applied Biosystems ViiA 7 Real Time PCR System can be powered off and stored for extended periods of time The length of the period of inactivity determines the method you use to power off the ViiA 7 Instrument Materials required MicroAmp Optical 96 384 Well Reaction Plate or array card unused Prepare the ViiA 7 Instrument 1 162 If you plan to store the ViiA 7 System for more than a week or you plan to move it load an unused plate or array card into the ViiA 7 Instrument Note The empty plate protects the internal components of the ViiA 7 System during transport or during periods of inactivity lasting more than a week a Touch the ViiA 7 Instrument touchscreen to awaken it then touch bi b Touch P to eject the tray arm place a plate or array card onto the plate adapter then press al again to load the plate c Touch 0 to place the ViiA 7 Instrument into stand by mode Toggle the power button on the rear of the ViiA 7 Instrument Power off the computer a Select Start gt Shut Down b In the Shut Down Windows dialog box select Shut Down then click OK Power off the monitor If you have an Applied Biosystems Twister II Robot toggle the power button on the rear of the Twister II Robot
157. e Note For the SDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical waste safety Chemical waste CAUTION HAZARDOUS WASTE Refer to Safety Data Sheets and local hazards regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles 232 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix F Safety F Chemical safety Chemical waste To minimize the hazards of chemical waste safety guidelines e Read and understand the Safety Data Sheets SDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste e Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible w
158. e 229 Calibration Maintenance Networking and Security F Appendix F Safety Instrumentation safety Safety and electromagnetic compatibility EMC standards U S and Canadian safety standards c UL us Canadian EMC standard European safety and CE standards Australia and New Zealand EMC standards 230 This section provides information on e U S and Canadian safety standards e Canadian EMC standard e European safety and EMC standards e Australia and New Zealand EMC standards The instrument has been tested to and complies with standard UL 61010 1 2nd Edition CSA C22 2 No 61010 1 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements UL 61010 2 010 Particular Requirements for Laboratory Equipment for the Heating of Materials This instrument has been tested to and complies with standard ICES 001 Issue 3 Industrial Scientific and Medical Radio Frequency Generators Cet appareil numerique de la classe B est conforme a la norme NMB 001 du Canada Safety This instrument meets European requirements for safety Low Voltage Directive 2006 95 EC This instrument has been tested to and complies with standards EN 61010 1 2001 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements EN 61010 2 010 2003 Particular Requirements for Laboratory Eq
159. e License keys are found on the ViiA 7 Software CD packaging or they can be supplied by Applied Biosystems support Manage licenses Monitor the current You can use the ViiA 7 Software to review the status and expiration date of the licenses licenses currently installed to the software 1 In the main menu of the ViiA 7 Software select Tools License Central 2 In the License Central dialog box review the status of your licenses The software displays the status of all installed licenses where possible states include Current and Expired and the date at which it expires Note The License Central dialog box lists the ViiA 7 Software core application and modules on different rows because the licenses are maintained separately 3 Optional If necessary save the license information to a text file a Select the license that you want to export from the table then click Save License Request Info b Navigate to the appropriate location then click Save 4 When you are done click OK Applied Biosystems ViiA 7 Real Time PCR System User Guide 133 Calibration Maintenance Networking and Security 5 Chapter 5 Service Manage ViiA 7 Software licenses Activate or renew a license Install a license file 134 If you have a valid licence key for the ViiA 7 Software or an associated module or if your license file has expired you can use the License Central feature to activate the license as explained
160. e 110 export records 114 export settings 120 import settings 120 object audit history 111 114 119 overview 101 purge records 113 restore records 113 system configuration history 111 112 114 119 when security is disabled 110 audit users enter reason for change 122 overview 121 Autodiscovery instrument 154 background calibration 48 242 data 49 perform 51 troubleshoot 53 when to perform 48 background fluorescence 49 253 Index backup experiment files 37 instrument settings 37 148 barcode file about 185 format 198 barcode readers 26 27 barcodes supported 26 baseline 242 baseline corrected normalized reporter DRn 242 biohazardous waste handling 234 biological replicate 242 blocked IPC 242 c calibration array cards 35 38 background 48 consumables 35 181 custom dye 168 dye 60 kits 177 normalization 69 plates 35 reminders enable disable 96 ROI 42 uniformity 55 calibrator 242 CAUTION description 14 chemical safety 231 chemical waste safety 232 233 clearances instrument components 21 required 21 command line application command syntax and arguments 188 189 running 187 compatibility third party software 31 computer experiment files maintenance 37 hard drives maintenance 37 remote monitoring 92 94 requirements 30 connections 23 consumables 181 contamination identification 54 contamination sample block decontamination 124 control instrument over a network 88 254 create array ca
161. e Cy values from the standard reactions plotted against standard quantities See also regression line e A set of standards containing a range of known quantities Results from the standard curve reactions are used to generate the standard curve The standard curve is defined by the number of points in the dilution series the number of standard replicates the starting quantity and the serial factor Method for determining absolute target quantity in samples With the standard curve method the software measures amplification of the target in samples and in a standard dilution series Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates the absolute quantity of target in the samples See also standard and standard curve In standard curve and relative standard curve experiments a set of standards containing a range of known quantities The standard dilution series is prepared by serially diluting standards For example the standard stock is used to prepare the first dilution point the first dilution point is used to prepare the second dilution point and so on In the software the volumes needed to prepare a standard dilution series are calculated by the number of dilution points the number of standard replicates the starting quantity the serial factor and the standard concentration in the stock See also standard curve In the PCR reaction a known quantity
162. e Ethernet cable to connect the Ethernet 1 port of the ViiA 7 Instrument to the nearest network port IMPORTANT Do not connect the Ethernet cable to the Ethernet 2 port on the ViiA 7 Instrument The second port is for Applied Biosystems use only 2 Power on the ViiA 7 Instrument 3 Use the ViiA 7 Instrument touchscreen to configure the network settings as described in Define the network settings on page 116 Applied Biosystems ViiA 7 Real Time PCR System User Guide 91 Calibration Maintenance Networking and Security 3 Chapter 3 Networking Connect the computer to the network Connect the computer to the network After connecting the ViiA 7 Instrument to the network connect the computer to the network and install the ViiA 7 Software for remote monitoring Materials required Ethernet cable with RJ45 connectors Computer requirement If you are connecting a computer that you provided to a network confirm that the computer contains a free network port Collect required information Obtain the following information from your network administrator e Network policy for obtaining IP addresses DHCP or static IP e Ifthe network requires static IP addresses obtain the IP address subnet mask and gateway address for the computer Set up the computer IMPORTANT We recommend that you arrange for a network administrator to connect your computer to the network The following procedure does no
163. e completed If electronic signature is enabled for experiments any of the following may apply e The Tools gt Security Sign Data menu option is enabled e You are prompted to sign as described in How the software prompts electronic signature on page 118 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security CHAPTER 5 Service This chapter covers m Decontaminate the sample block 66 c cece 124 E Replace the halogen lamp 1 6 6 6 ccc ee eens 127 m Replace the instrument fuses 0 6 eee nes 130 m Update the Windows operating system 0 66 e cece ee eee ees 131 m Update the ViiA 7 Software and Firmware 000 0c cece eee ees 132 m Manage ViiA 7 Software licenses 0 6 133 E Replace the sample block 0 cece eee nee 135 mM Replace the heated cover 6 6 eee eee eens 137 E Replace the plate adapter eens 139 IMPORTANT This chapter contains all user service procedures for the Applied Biosystems ViiA 7 Real Time PCR System Procedures other than those described in this document must be performed by a qualified Applied Biosystems service engineer Applied Biosystems ViiA 7 Real Time PCR System User Guide 123 Calibration Maintenance Networking and Security 5 Chapter 5 Service Decontaminate the sample block Decontaminate the sample block Materials required How to
164. e computer ordered with the ViiA 7 System laptop or desktop The Applied Biosystems Twister II Robot is an optional component of the ViiA 7 System Required clearance The ViiA 7 Instrument requires the following additional clearances e Clearance on all sides At least 15 2 cm 6 in of clearance for ventilation service access and cable routing e Vertical clearance At least 30 5 cm 12 in of unobstructed vertical clearance above the ViiA 7 Instrument to allow removal of the cover during service Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 21 1 Chapter 1 Getting Started Specifications and layout Instrument hot air The maximum thermal output of the ViiA 7 Instrument is 2731BTU hr 800W vented exhaust venting directly into the room air from the hot air waste port on the rear panel Electrical Note We recommend placing the ViiA 7 Instrument and computer power receptacle requirements on an electrical circuit that is not shared with electrically noisy devices or devices that can cause power surges such as refrigeration units The following table provides electrical specifications for the instrument and associated devices For all indicated input voltages a 15 A circuit is required Device Rated Rated Rated Rated current A power VA voltage VAC frequency Hz Instrument 12 5 950 Computer Desktop 2
165. e eee ene ees 154 Define the system shortcuts 0 6 ccc ccc E enn e nn ees 155 Review the instrument statistics 0 eee eee eee 155 Enable or disable instrument security 66 156 View the instrument log sise 2 eee enn eens 157 Note This appendix describes how to operate the ViiA 7 Instrument manually using the touchscreen interface Although the ViiA 7 Instrument can be used without a physical attachment to a computer the touchscreen allows you to perform only a subset of the total instrument functions Applied Biosystems ViiA 7 Real Time PCR System User Guide 141 Calibration Maintenance Networking and Security A Appendix A Manual Instrument Operation Overview Overview The ViiA 7 Instrument features a touchscreen interface that you can use to run experiments manage instrument settings and configure the ViiA 7 Instrument for network use The touchscreen does not provide access to all instrument functions Features such as experiment analysis instrument calibration and remote notification are available only through the ViiA 7 Software Functions available from the instrument touchscreen 142 The following table summarizes the functions that are available from the ViiA 7 Instrument touchscreen The table organizes the functions by user role where operational functions are for users that perform experiments maintenance functions are for users who maintain the instrument and
166. e experiment will use e The name of a dye in the Dye Reference as a passive reference Library of the ViiA 7 Softwaret or e lt blank gt if the consumable does not contain a passive reference Custom dyes are allowed as long as they are in Dye Library Note The ViiA 7 Software automatically removes any leading and trailing white space around the field name and field value Example Instrument Type ViiA 7 Passive Reference ROX Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference E Import formats and file specifications Plate setup file body The body of a plate setup file contains either target information which can be imported into all experiments except genotyping or SNP assay information This information can be imported into genotyping experiments only The body consists of three required elements the header the column header and the body that describe the contents of a 96 384 well plate or array card The sample setup column header and body can appear in any order IMPORTANT Observe the following guidelines when creating a plate setup file e Do not insert blank lines between the sample setup header and the column header e Do not use illegal characters including backslash tab asterisk hard return soft return brackets or or comma Sample setup header The header contai
167. e is at the bottom of the well If not centrifuge the plate again at a higher rpm and fora longer period of time Correct Incorrect Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the uniformity calibration Perform the calibration 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a ViiA 7 Instrument to your list before you can manage it 3 After the ViiA 7 Instrument is added to your list of instruments select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenance then click Uniformity b In the Uniformity Calibration screen click Start Calibration 5 Click Next then perform the calibration as instructed When the side door opens load the ROI calibration plate or array card Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the bar code facing the front of the instrument IMPO
168. eal Time PCR System User Guide Calibration Maintenance Networking and Security Contents CHAPTER 5 Service sos ect itmsd deere eel eO ole eee a E 123 Decontaminate the sample block 2060 cece eee eee e eens 124 Materials required 020 ccc ccc eee ence eee aee 124 How to handle the sample block 0c cece eee eee eee teens 124 Clean the sample block 00 ccc eee eee ranana nnne 125 Replace the halogen lamp 2 nnn ce rnrn eee e eee e eens 127 Materials required 020 e eee eens 127 Halogen lamp warningS 2 ee cece eee eee eee eee ence eee e eens 127 Check the lamp status 0000 c cece cece teen eeee 128 Replace the lamp 02 cece eect eee eee e eens 128 Replace the instrument fuses 2 00 cece cece eee eens 130 Materials required 020 c cece ccc eee eee e eens 130 Replace the fuses 0202 e eee eens 130 Update the Windows operating system 0 ccc ccc nce e nnr eee eee nes 131 Update the ViiA 7 Software and Firmware 0 0c cece cece cece cece cena ees 132 Update the ViiA 7 Software 0 ccc cnc ene nt e ene eenes 132 Update the ViiA 7 Instrument firmware 0 cece cece nee n ene en ee 132 Manage ViiA 7 Software licenses 0 kc nce nnn rrer 133 About ViiA 7 Software license keys and files 0 00 cece ec cece eee eee 133 Manage licenses
169. ect from the packing box carefully tilt the box on its side and hold it stationary while someone slides the contents out of the box Ensure that anyone who operates the instrument has instrument e Received instructions in both general safety practices for laboratories and specific 8 yP P safety practices for the instrument e Read and understood all applicable Safety Data Sheets SDSs See About SDSs on page 232 Cleaning or CAUTION Before using a cleaning or decontamination method other than decontaminating the those recommended by the manufacturer verify with the manufacturer that the instrument proposed method will not damage the equipment Applied Biosystems ViiA 7 Real Time PCR System User Guide 227 Calibration Maintenance Networking and Security F Appendix F Safety Instrumentation safety Physical hazard sa Ultraviolet light Moving parts Electrical safety Fuses Power Overvoltage rating 228 fety AN WARNING ULTRAVIOLET LIGHT HAZARD Looking directly at a UV light source can cause serious eye damage Never look directly at a UV light source and always prevent others from UV exposure Follow the manufacturer s recommendations for appropriate protective eyewear and clothing WARNING PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing the instrument WARNING ELECTRICAL SHOCK
170. ed data when computing variability estimates of the single biological replicate Individual contributions of the separate biological samples to the single biological replicate results are observed in the Technical Replicates tab See also technical replicates In presence absence experiments a reaction that contains IPC blocking agent which blocks amplification of the internal positive control IPC In ViiA 7 Software also the name of the task for the IPC target in wells that contain IPC blocking agent See also negative control blocked IPC wells See reference sample See reagents See threshold cycle CT Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security custom dye cycle threshold cycling stage delta Rn ARn dye calibration Glossary Dye that is not precalibrated for an instrument Custom dyes that fall within the emission wavelength range of the instrument can be added and adapted for use in experiments on the ViiA 7 Instrument To use a custom dye add the dye to the Dye Library and perform a dye calibration See threshold cycle CT In the thermal profile a stage that is repeated A cycling stage is also called an amplification stage See also amplification stage See baseline corrected normalized reporter DRn Type of calibration in which the software collects spectral data from a series of dye standards and stores the spectral in
171. eee 55 Matertals required n saine ipi naa et aces glee na ane BARES GREE we 55 When to perform the calibration 200cc cece eee eee eens 55 About the uniformity calibration data 000s 55 Prepare the calibration plate or array card 20 cece eee eee eeee 56 Perform the calibration 0 cece cece eee eee eee eee eeaas 57 Troubleshoot the uniformity calibration 0000 cece eee eee 59 Perform the dye calibration 0 000 cece cece annarar erann 60 Materials required 20 uote Ait ee lid ee Mie lid ee eee os Lele A 60 When to perform the dye calibrations 2000c cece eee eee eee 61 About the dye calibration E A e eeee 62 Prepare the calibration plates array cards unninn cece eee eee eens 64 Perform the calibration 0 rea eee tenet tenet eens 65 Troubleshoot the dye calibration 0 00 cece eee eee eee eens 68 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Contents Perform the normalization calibration 0 00 c cece eee cnet nets 69 Materials required nnana cece ett e ee eee eee eee 69 When to perform the calibration 20000 nrnna rnnr eee eee 69 About the normalization calibration data ccc cece eee eee eee 69 Prepare the calibration plate or array card sunnan uann eee eee e eee eee 70 Perform the cal
172. ells of the target sample combination RQ Min The minimum relative quantity calculated for the Float replicate wells of the target sample combination The lower limit of the confidence interval RQ Max The maximum relative quantity calculated for the Float replicate wells of the target sample combination The upper limit of the confidence interval Ct The calculated threshold cycle C7 for the target at Float the specified well Ct Mean The average C of the replicate wells for the Float specified target sample combination Delta Ct The AC value of the replicate wells for the Float specified target sample combination Delta Ct Mean The average AC of the replicate wells for the Float specified target sample combination Delta Ct SD The standard deviation of the AC for the replicate Float well Depending on the analysis settings this column may by replaced with Delta Ct SE the standard error of the AC Delta Delta Ct The AAC value of the replicate wells for the Float specified target sample combination Automatic Ct Indicates whether the threshold was determined true Or false Threshold automatically true or manually false Ct Threshold The threshold cycle C7 for the sample at the well Float Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 213 Appendix E ViiA 7 Software Reference Export formats and file specifications Column Description Out
173. elow Note See the Applied Biosystems ViiA 7 Real Time PCR System Robotics Guide PN 4442663 for information on operating calibrating maintaining and integrating the Twister II Robot Component Description Reach axis Moves the grip horizontally 11 25 in 28 5 cm to 19 75 in 50 1 cm from the center of the robot post Wrist Rotates materials to either the portrait or landscape positions where mechanism the range of motion is 135 270 total Grip Consists of two sets of fingers that grip the consumable The fingers close to grasp a consumable and open to release it Robot tower Moves the arm up and down 21 5 in 54 6 cm from 6 5 in 16 5 cm to vertical axis 28 in 71 1 cm above the table Rotary axis Rotates the arm 340 around the base of the Twister II Robot Mechanical stops prevent continuous rotation Bar code Scans the bar codes of consumables as they are loaded into the ViiA 7 reader Instrument Base cover Removable cover that contains four access bolts which secure the Twister II Robot to the Sciclone ALH 3000 base Racks Provide storage for PCR consumables before and after they are run by the ViiA 7 Instrument one of three shown Power LED When lit indicates the Twister II Robot is powered on 27 1 Chapter 1 Getting Started ViiA 7 System hardware Rear view Comp
174. em LIMS applications or databases that have been configured to parse the file format The following table shows the data structure common to data exported in the ViiA 7 export format regardless of experiment type Each row represents one or more lines of data in the exported file corresponding to a common functional group Because the ViiA 7 export format allows the user to customize and or omit columns The columns and orders described below are the default configuration all columns in their natural order Actual files may contain fewer columns if the user modified the configuration Section Description See File header Describes the qualities of the ViiA 7 Instrument used to page 201 run the experiment and several general experiment properties such as the date and time of the run and the dye used as the passive reference Sample setup Describes the configuration of samples on the experiment page 202 data consumable including sample location target or SNP assay properties and task assignments Raw data Contains the raw data collected by the ViiA 7 Instrument page 204 during the experiment run Amplification Contains the normalized data collected during the cycling page 205 data stage of PCR amplification which the ViiA 7 Software uses to generate the amplification plot Note Not applicable for presence absence genotyping or melting curve experiments that are run without a PCR cycling stage
175. ement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Instrument malfunction Multiple possible causes Contact a local Applied Biosystems Field Service Office Applied Biosystems ViiA 7 Real Time PCR System User Guide 85 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Verify the instrument performance 86 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security This chapter covers E Networking overview 0 0c eee e eee m Network setup workflow 04 m Connect the ViiA 7 Instrument to the network m Connect the computer to the network E Monitor the ViiA 7 Instrument CHAPTER 3 Networking Access the Help system by pressing F1 by clicking in the toolbar of the ViiA 7 Software window or by selecting Help gt Contents and Index IMPORTANT This chapter does not provide adequate detail to integrate the Applied Biosystems ViiA 7 Real Time PCR System into all possible network architectures Because your network may contain advanced features such as a firewall or network domains we recommend that you consult a network administrator before connecting the ViiA 7 System to your laboratory network Applied Biosystems ViiA 7 Real Time PCR System
176. ered from the Applied Biosystems website Use the consumables appropriate for the sample block of your ViiA 7 System Sample Consumable Reaction block volume 96 well e MicroAmp Optical 8 Cap 50 uL plate Strip 0 2 mL e MicroAmp 8 Tube Strips 0 2 mL e MicroAmp Reaction Tubes without Caps 0 2 mL e MicroAmp 96 Well Tray Retainer Set e MicroAmp Optical 50 uL Adhesive Film e MicroAmp Optical 96 Well Reaction Plate with Bar Code 96 well e MicroAmp Optical 50 uL plate Adhesive Film 0 1 mL e MicroAmp Optical 96 Well Fast Reaction Plate with Bar Code 384 well e MicroAmp Optical 20 uL plate Adhesive Film e MicroAmp Optical 384 Well Reaction Plate with Bar Code Array card Applied Biosystems Array Card Tul Applied Biosystems ViiA 7 Real Time PCR System User Guide 35 Calibration Maintenance Networking and Security 36 Chapter 2 Calibration and Maintenance About the consumables Observe the following guidelines when using tubes plates or array cards Store the calibration plates or array cards in a dark place until you are ready to use them The fluorescent dyes in the wells of calibration consumables are photosensitive Prolonged exposure to light can diminish the fluorescence of the dyes Do not allow the bottom of the tubes plates or array cards to become dirty Fluids and other contaminants that adhere to the bottoms of the consumables can contaminate the samp
177. eriment from a template 0 6 00 cece eee eee 143 E Runan experiment sici euer tea sai e eal eee ano da eee 144 m Transfer experiments templates and results data 000005 145 Note If the ViiA 7 Instrument is operating in secure mode see Enable or disable instrument security on page 156 users can only open and close the side door Create an experiment from a template 1 If necessary download the experiment template to the ViiA 7 Instrument as described in Transfer experiments from a USB drive on page 145 2 If the instrument is in standby touch the ViiA 7 Instrument touchscreen to awaken it then press Ot 3 In the Main Menu touch F View Templates 4 In the View Templates screen create the experiment a Touch then touch the folder that contains the desired template b Touch the desired template c Touch wf Create Experiment To view the parameters of a template select the desired template then touch 18 View When finished touch 4 to return to the View Templates screen Note You cannot modify the experiment parameters of a template 5 In the Create New Experiment screen touch each field to set the e Experiment name e Folder to receive the experiment e Reaction volume e Bar code e Any additional information to save to the experiment 6 When finished either e Touch Save amp Exit then touch to return to the Main Menu or e Touch Save amp Start Run to
178. es 2 Open the access door Applied Biosystems ViiA 7 Real Time PCR System User Guide 137 Calibration Maintenance Networking and Security 138 Chapter5 Service Replace the heated cover 10 Unlock the heated cover by pinching the handle together then pull the assembly from the ViiA 7 Instrument and place it on a clean dry surface Install the new heated cover into the ViiA 7 Instrument IMPORTANT When the heated cover is seated correctly the arrows on the front handle align as shown below If the arrows do not align push the heated cover further into the ViiA 7 Instrument until the handle locks into place Correct Incorrect If you are installing a heated cover of a different consumable format replace the sample block and plate adapter if necessary IMPORTANT If you are installing a heated cover of a different format you must also change the sample block and plate adapter to match the new consumable format Close the access door Confirm that the access door is completely closed The ViiA 7 Software displays an error message if the door is not completely closed and latched or if the sample block is not seated correctly Plug in and power on the ViiA 7 System In the Home screen of the ViiA 7 Software click Instrument Console In the Instrument Console select your ViiA 7 Instrument from the list of instruments then review the Heated Cover Firmware
179. es the Analysis Status check but fails the QC Status check We recommend using calibrations that yield passing results for both status reports Failed Failed WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can be heated to 100 C Before removing the plate wait until it reaches room temperature IMPORTANT If the ViiA 7 Instrument does not eject the plate remove the plate as explained in Troubleshoot the dye calibration on page 68 10 Discard or store the plate or array card Consumable Action Array card Discard the array card Plate Return the dye calibration plate to its packaging sleeve then return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate is stored in its original packaging sleeve you can use it to calibrate a ViiA 7 System 3 times for up to 6 months after you open it 11 Repeat the calibration and review step 4 through step 10 to calibrate your ViiA 7 System for the remaining dyes that are present in the chemistries that you will be running Applied Biosystems ViiA 7 Real Time PCR System User Guide 67 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the dye calibration Troubleshoot the dye calibration Problem symptom Possible cause Action One or more raw spectra are at or below the detectable threshold for the calibra
180. esults Reviewed and Approved Plate Set Up Yes Yes No No Reviewed and Approved Results Yes Yes Yes Yes Reviewed and Approved Template Yes Yes No No Add a meaning 1 In the e Signature tab of the Security Settings dialog box click Add in the e Signature Meanings settings 2 In the Create Meaning dialog box enter a description of the e Signature meaning then click OK 3 Select what data is signed for the selected meaning 4 Click Apply Settings 116 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators 4 Manage electronic signature Delete a meaning 1 Select the meaning from the e Signature Meanings list then click Remove 2 Click Apply Settings Configure the e signature rights for user roles To determine the user roles that can perform an electronic signature 1 In the e Signature tab of the Security Settings dialog box select the check box next to the appropriate user roles in the User Role signature rights table 2 Click Apply Settings Select the actions that require signature IMPORTANT Do not change electronic signature settings during calibration 1 In the Signature Required column select the check box next to each action for which you want to require electronic signatures see below This selection causes the software to present an e sig prompt if a user performs the action on a data file that does not have the requi
181. fication cycle Ca quantity quencher R2 value raw data plot reaction mix reagents real time PCR Real time PCR Data Markup Language RDML reference reference sample refSNP ID Glossary The fractional PCR cycle used for quantification according to the Real time PCR Data Markup Language RDML data standard CT and CRT are the algorithm specific calculations of C In quantification experiments the amount of target in the samples Absolute quantity can refer to copy number mass molarity or viral load Relative quantity refers to the fold difference between normalized quantity of target in the sample and normalized quantity of target in the reference sample A molecule attached to the 3 end of TaqMan probes to prevent the reporter from emitting fluorescence signal while the probe is intact With TaqMan reagents a nonfluorescent quencher minor groove binder NFQ MGB can be used as the quencher With SYBR Green reagents no probe and therefore no quencher is used Regression coefficient calculated from the regression line in the standard curve An important quality value the R value indicates the closeness of fit between the standard curve regression line and the individual Cy data points from the standard reactions A value of 1 00 indicates a perfect fit between the regression line and the data points A plot of raw fluorescent signal as detected through each emission filter used to view raw data
182. figured to suspend a user account for failed logins and you enter an incorrect user name and password for more than the allowed number of times your user account is suspended and the Log In dialog box indicates that your account is inactive There are two ways to activate a suspended account e You can wait until the suspension period ends e An administrator can change the account status from Suspended to Active Note While a user is suspended another user can click Reset then log in and replace the suspended user If the ViiA 7 Software is configured to time out and there is no user activity for the specified time the Log In dialog box indicates that your user session has timed out You must enter your user name and password to access the software The administrator or another user with permission to log in to timed out sessions can click Reset then log in If the ViiA 7 Software is configured for auditing you may be prompted to specify a reason when you make certain changes in the software Depending on your ViiA 7 Software configuration you can either select a reason from the list or enter a reason for change Electronic signature 122 If your system is configured for electronic signature you may be required to have the experiment signed by other users before you can print a report or start a run If anitem is set to require multiple signatures all approvers must sign the associated data before the action can b
183. for each point in the standard curve For example if the standard curve is defined with a serial factor of 1 10 or 10X the difference between any 2 adjacent points in the curve is 10 fold Regression coefficient calculated from the regression line in the standard curve The slope indicates the PCR amplification efficiency for the assay A slope of 3 32 indicates 100 amplification efficiency See also amplification efficiency EFF and regression line In the thermal profile a group of one or more steps Examples PCR stage cycling stage also called amplification stage and hold stage A sample that you dilute and amplify along with unknown samples This dilution series can contain known starting quantities of the target of interest absolute standard curve or it can be of known dilution factor relative standard curve Following the run the software interpolates the Cr values of the unknowns to this curve yielding either specific quantities of the target for absolute curves or relative quantities for relative dilution curves See also standard curve Applied Biosystems ViiA 7 Real Time PCR System User Guide 249 Calibration Maintenance Networking and Security Glossary standard curve standard curve method standard dilution series standard quantity starting quantity step SYBR Green reagents 250 In standard curve and relative standard curve experiments e The best fit line in a plot of th
184. for the instrument run The run method specifies the temperature time ramp and data collection points for all steps and stages of the instrument run The biological tissue or specimen that you are testing for a target gene A tab delimited file txt or csv that contains the following setup information well number sample name and custom sample properties An optional software module that provides e System Security Controls user access to the software A default Administrator user account is provided and you can define additional user accounts and permissions e Auditing Tracks changes made to library items actions performed by users and changes to the Security and Audit settings The software automatically audits some actions silently You can select other items for auditing and specify the audit mode Provides reports for audited library items Security and Audit changes and actions e Electronic Signature eSignature Controls whether users are permitted prompted or required to provide a user name and password when accessing certain software features You can select which features are controlled and the number of signatures required for access When authorized persons use this feature they are creating a legally binding signature In the software a numeric value that defines the sequence of quantities in the standard curve The serial factor and the starting quantity are used to calculate the standard quantity
185. for the move a Open the ViiA 7 System access door b Firmly press down on the sample block handle pull the sample block from the ViiA 7 Instrument then place it on a clean dry surface c Pinch the handle of the heated cover together then pull the assembly from the ViiA 7 Instrument and place it on a clean dry surface Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 163 Appendix B Power On or Off Store and Move the ViiA 7 System B Move the ViiA 7 System d Package the sample block and heated cover assemblies in a clean dust free container for the move Move the ViiA 7 System Move the ViiA 7 System according to the following guidelines Verify that the surface on which you will place the ViiA 7 System can support at least 60 140 6 kg 132 5 0 13 Ibs Verify that the path to transport the ViiA 7 Instrument is clear of obstructions Enlist at least one other person to lift and carry the ViiA 7 Instrument Keep your spine in a good neutral position Bend at the knees and lift with your legs Do not lift an object and twist your torso at the same time Coordinate your intentions with your assistant before lifting and carrying Reinstall the ViiA 7 System 1 164 Reconnect the components of the ViiA 7 System Use the Ethernet cable supplied with the ViiA 7 System to connect the ViiA 7 Instrument E
186. for the wells should be similar Variations in spectral position and peak position are caused by minor differences in the optical properties and excitation energy between the individual wells Acceptable spectra Unacceptable spectra Spectra peak at the same wavelength and do Spectra peak at the different wavelengths not diverge significantly j O i C M Normalized Fluorescence Normalized Fluorescence PHHH n L nn _____1AW Ly oo ie bd 21 tneft m t m 1 4 miB m 2 m 2 mi m mei2 o63 mB rre m6S MOt rrad4 m mBS mi66 meB m6 2c 1 2 8 rmal m 1 mE2 m22 m82 m2 m2 m83 MB M43 m53 MeH m44 m6 m85 m 5 mB M8 Filter Filter Applied Biosystems ViiA 7 Real Time PCR System User Guide 63 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the dye calibration Prepare the calibration plates array cards Prepare the dye calibration plate 64 IMPORTANT Before performing a dye calibration you must perform an ROI calibration a background calibration and a uniformity calibration IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or array cards Prepare the Dye calibration consumables appropriate for your ViiA 7 Instrument Prepare the dye calibration plate 0 0 see below Fill the array Cards ince ines pod ernea bodes tha dp ase onde i a E 38 Remove the dye plates from the freezer then allow
187. formation for the dye standards in a pure spectra calibration file This file is used during experiment runs to characterize and distinguish the individual contribution of each dye in the total fluorescence collected by the instrument Dye Library In the software a collection of dyes to use in experiments Custom dyes can be added to the library but system dyes cannot be removed Before using a dye make sure that the dye calibration is current in the Instrument Console EFF See amplification efficiency EFF error The standard error of the slope of the regression line in the standard curve The error can be used to calculate a confidence interval CI for the slope Because the amplification efficiency EFF is calculated from the slope knowing the error allows a CI for the amplification efficiency to be calculated experiment Refers to the entire process of performing a run including setup run and analysis You can perform the following types of experiments e Quantification Standard curve e Quantification Relative standard curve e Quantification Comparative Cy AACy e Melt Curve e Genotyping e Presence absence experiment The Applied Biosystems name for the electronic records that comprise all information document about a particular plate or array card consumable including metadata name bar code comments plate setup well contents assay definitions run method thermal cycling protocol run results analysis protocol
188. hange the instrument name or IP address Note To reestablish the connection restart the ViiA 7 Software 94 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 3 Networking 3 Monitor the ViiA 7 Instrument Upload or download an experiment or template to a ViiA 7 Instrument Note The ViiA 7 Instrument can store up to 100 gene expression experiments Before sending an experiment confirm that the instrument contains sufficient storage space 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a ViiA 7 Instrument to your list before you can manage it 3 After the ViiA 7 Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager click Manage Files then click File Manager 5 In the File Manager screen transfer the file s To upload a file to the ViiA 7 Instrument a In the Folders field select the folder to which you want to upload the file To create a new folder click Create then enter a name for the new folder b Click Upload select the experiment or template file to send to the ViiA 7 Instrument then click Open TM To download a file from the ViiA 7 Instrument a In the Folders field sele
189. he adhesive cover 1 Power off the ViiA 7 Instrument eject the ROI plate may have adhered 2 Wait for 15 minutes then power on the ViiA 7 Instrument and eject the plate to the the plate heated cover within a the instrument 3 If the plate does not eject power off and unplug the ViiA 7 Instrument then open the access door 4 Wearing powder free gloves reach into the ViiA 7 Instrument and remove the plate from the heated cover then close the access door 5 Perform a background calibration to confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a local Applied Biosystems Field Service Office Applied Biosystems ViiA 7 Real Time PCR System User Guide 47 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Perform the background calibration Perform the background calibration During a background calibration the ViiA 7 System Performs reads of a background plate containing PCR buffer for 10 minutes at 60 C Averages the spectra recorded during the run and extracts the resulting spectral component to a calibration file The ViiA 7 Software then uses the calibration file during subsequent runs to remove background fluorescence from the run data Materials required 96 Well Plate E Sample Block 96 Well Region of Interest ROI and Background Plates Centrifuge with plate adapter Powde
190. he experiment lt 100 character string gt Experiment File Name The path to the experiment file on the local computer hard drive lt filepath gt Experiment Name The name of experiment entered into the Experiment Name field lt 100 character string gt Experiment Run End Time The date and time that the ViiA 7 Instrument finished running the experiment lt date and time gt Experiment Type The type of chemistry application for which the experiment is designed Standard Curve Presence Absence Relative Standard Curve Or DDCt Quantification Instrument Type The model of the ViiA 7 Instrument that ran the experiment ViiA 7 Passive Reference The dye used as a passive reference or blank if the consumable did not contain one lt 100 character string gt Signal Smoothing On The smoothing setting status for the experiment Indicates whether smoothing is turned on for the experiment true OF false Stage Cycle where Analysis is performed The stage and cycle during the thermal cycling protocol when the ViiA 7 Instrument collected data Stage lt integer gt Step lt integer gt Calibration Date Calibration Expiration Date The date and time that the current background ROI uniformity or pure dye calibration was performed and when it will expire lt date and time gt lt date and time gt Instrument serial number The ser
191. he experiment run The raw data consists of fluorescence readings collected by the ViiA 7 Instrument that have not been normalized to the passive reference The section begins with a column header followed by the raw data where each row contains the data for a single well separated by tab characters Each line of raw data consists of readings sorted by bin where each bin represents an excitation emission filter pair that was selected during experiment setup The bins are named for the corresponding filter combination according to the following convention lt excitation filter name gt lt emission filter name gt Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 t Cycle The cycle of the run during which the ViiA 7 Integer Instrument recorded the fluorescence lt Bin gt The raw fluorescence for the well measured by Float the ViiA 7 Instrument for the associated bin at the designated cycle Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom 204 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Amplification data Multicomponent data Appendix E ViiA 7 Software Reference Export formats and file specifications The ViiA 7 Software can export the processed amplification data used to generate the amplification plot of a real time
192. he headings and columns that are present in the plate setup file body of all experiment types followed by the columns that are specific to genotyping experiments and non genotyping experiments Column name Description Valid values Well The number of the well on the consumable where the well numbers start at 1 for well A1 upper left corner and increase lt Positive integer 1 to 96 384 gt 8 Allele1 Quencher The quencher dye used to label the probe for the first allele a from left to right and from top to bottom Sample Name The name of the sample contained by the associated well lt 100 character string gt A Sample Color Optional The RGB color of the associated sample RGB lt r gt lt g gt lt b gt x Biogroup Name Optional The name of the associated biological group lt 100 character string gt Biogroup Color Optional The RGB color of the biological group RGB lt r gt lt g gt lt b gt Comments Optional Additional text that describes the well lt 1024 character string gt Target Name The name of the target detected or amplified by the assay in lt 100 character string gt tt the associated well Target Color Optional The RGB color of the target RGB lt r gt lt g gt lt b gt Task The task assignment of the target assay at the well t lt UNKNOWN STANDARD NTC ENDOGENOUS S IPC BlockedI
193. hich when applied by the software to a well during analysis indicates a possible issue with that reaction For example a flag may be issued if no amplification is detected in a well Flags indicating potential problems are displayed in the Quality Control tab of the plate layout well table and QC Summary screens Oligonucleotide that flanks the 5 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target In the thermal profile the stage that holds the temperature constant for a defined period of time A stage that includes one or more steps You can add a holding stage to the thermal profile to activate enzymes to inactivate enzymes or to incubate a reaction A gene that is involved in basic cellular functions and that may be constitutively expressed Housekeeping genes may be candidates for use as endogenous controls however their constancy should always be validated experimentally See also endogenous control A software feature that allows you to import plate setup information or security settings before an experiment run You can also import information into some libraries in the system Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Instrument Console Instrument Manager internal positive control IPC IPC IPC melt curve stage melting temperature Tm melting transition region
194. holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the bar code facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 6 After loading the plate or array card start the calibration a In the Setup tab select Check the box when the ROI calibration plate has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the ViiA 7 Instrument is in operation Note Before starting the calibration the ViiA 7 Instrument may pause up to 10 minutes to allow the heated cover to reach temperature 7 When the run is complete and the Analysis screen displays select each filter from the Filter Set drop down list then verify that the corresponding ROI Image displays a green circle around each well area Applied Biosystems ViiA 7 Real Time PCR System User Guide 45 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the ROI calibration 8 After you inspect all ROI images verify the status of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or that the data it did col
195. ial number of the ViiA 7 Instrument that ran the experiment lt 100 character string gt Quantification cycle method The method of quantification for the associated experiment lt 100 character string gt Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 201 Sample setup data 202 Appendix E ViiA 7 Software Reference Export formats and file specifications When selected as an export option the ViiA 7 Software exports sample setup data after the file header The sample setup data describes the sample configuration on the experiment consumable including positions sample names task assignments assay information and color coding The data consists of a column header followed by the sample data fields where each row contains the data for a single well separated by tab characters If a well contains more than one assay target the ViiA 7 Software lists the data for each additional assay on separate rows repeating the well number and sample information The data included in the sample setup data export varies depending on experiment type This section describes the following sample setup data formats Quantification and presence absence experiments 000 rnrn 202 E Genotyping experiments 6 6 eee tenes 203 Quantification and presence absence experiments The table below describes the sample setup data that can be
196. ibration s se 2 ccceuete tore caeh pecan cts eo oe tee e ad sted tay ae ads 71 Troubleshoot the normalization calibration 0 0 cece eee eee eee 73 Verify the instrument performance 22000 0 cece eee eee eee eee e eee eaes 74 Materials required 0 0 e cece ccc eee eee eee eaee 74 When to perform the RNase P instrument verification experiment 74 About the RNase P kits 0 0 eee een ene eee enn ERRA 75 Aboutthe analysise 4 3 24 oco0 5 ee Sale od ah Sane ae Da et ch at ON cree at 76 Installation specification s 2c2252s4 ences e dasa goed Menges then ges eed eget 76 Prepare the TaqMan RNase P plate or array card 0 cece eee es 77 Run the experiment sasssa derino ea ee peed ea AANA see ables WG kena a tienneale 79 Troubleshoot the RNase P experiment 0000 eee eee cece 83 CHAPTER 3 Networking cece cc cuieiee eee eo ot Sela eee alodewee sunt votes 87 Networking overvieW 000 ce cece cece ete eee tect eee eee 88 Controlling and monitoring networked ViiA 7 Instruments 00005 88 About the Ethernet 1 port 22 2202 25 eee eee eee ee eens 88 Example network layouts 000 e cece eee e eens 89 Networking guidelines and best practices 200ce eee eee eee 90 Network setup workflow 002 cece cece nrnna nanna 90 Collect the required network information 0200 cece cece eee ee eee 91
197. ils again contact Applied Biosystems technical support Instrument does not eject the normalization plate The adhesive cover may have adhered the plate to the heated cover within the instrument 1 Power off the ViiA 7 Instrument 2 Wait for 15 minutes then power on the ViiA 7 Instrument and eject the plate 3 If the plate does not eject power off and unplug the ViiA 7 Instrument then open the access door 4 Wearing powder free gloves reach into the ViiA 7 Instrument and remove the plate from the heated cover then close the access door 5 Perform a background calibration to confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a local Applied Biosystems Field Service Office Applied Biosystems ViiA 7 Real Time PCR System User Guide 73 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Verify the instrument performance Verify the instrument performance Perform the RNase P instrument verification experiment to verify the performance of an Applied Biosystems ViiA 7 Real Time PCR System Materials required 96 Well Plate e Centrifuge with plate adapter Sample Block e Powder free gloves e Safety goggles e TaqMan RNase P Fast 96 Well Instrument Verification Plate 384 Well Plate e Centrifuge with plate adapter Sample Block e Powder free gloves e Safety goggles e
198. iments See Assay information file on page 198 for more information barcode file txt A user created line separated text file that contains the bar code of each consumable for which you want to create an experiment file See Bar code file format on page 198 for more information experiment A ViiA 7 Software file that contains all information about a particular document single plate or array card consumable including metadata name bar code file eds comments plate setup well contents assay definitions run method thermal cycling protocol run results analysis protocol analysis results audit records and other plate specific data experiment A ViiA 7 Software file used as a template to create experiment files document The file can contain plate setup well contents assay definitions run template file edt method thermal cycling protocol run results analysis protocol and other plate specific data plate setup file txt A user created tab delimited text file that describes the layout of a consumable for an experiment to be run on the ViiA 7 System The file defines the arrangement of assays and samples on the consumable See Plate setup file format on page 192 for more information sample file txt A user created tab delimited text file containing sample data that can be imported into the ViiA 7 Software for use in related experiments See
199. ion and RNase P Fast 96 quantitation of genomic copies of the human RNase P gene a single copy gene 384 Well Instrument encoding the RNase moiety of the RNase P enzyme Each well contains TaqMan Fast Universal PCR Master Mix RNase P primers FAM dye labeled probe and a Verification Plate known concentration of human genomic DNA template The figure to the right illustrates the arrangement of the standard and unknown populations on a 96 well Unknown A RNase P plate The RNase P plate 5000 contains five replicate groups of standards 1250 2500 5000 10000 and 20000 copies two unknown populations 5000 and 10 000 copies and ano template control NTC Unknown B 10000 The figure to the right illustrates the arrangement of the standard and unknown populations on a 384 well RNase P plate The RNase P plate contains five replicate groups of standards 1250 2500 5000 10000 and 20000 copies two unknown populations 5000 and 10 000 copies and ano template control NTC Unknown A Unknown B 5000 10000 p Standard 1250 5 Standard 2500 ine p Standard 5000 Standard 10000 P Standard 20000 e About the Array Card The Array Card RNase P Kit includes one empty array card and eight tubes of RNase P Kit solution Each tube contains reaction mix TaqMan Universal PCR Master Mix RNase P primers and FAM MGB dye labeled probe and a known concentration of human genomic DNA templa
200. ircles appear around some or none of the wells indicating that the wells did not calibrate The absence of a circle indicates that the region of interest for the well position is not sufficiently bright Prepare the calibration plate or array card IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or array cards Prepare the ROI calibration consumable appropriate for your ViiA 7 Instrument m Prepare the ROI calibration plate 2 6 44 M Filthearray cards vhs scstieeiiee dates swe E eee eae ks 38 Applied Biosystems ViiA 7 Real Time PCR System User Guide 43 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Perform the ROI calibration Prepare the ROI 1 Remove the ROI calibration plate from the freezer then allow it to warm to room calibration plate temperature approximately 5 minutes IMPORTANT Do not remove the calibration plate from its packaging until you are ready to run it The fluorescent dyes in the wells of the plate are photosensitive Prolonged exposure to light can diminish the fluorescence of the dye 2 Remove the ROI calibration plate from its packaging Do not remove the optical film IMPORTANT Do not discard the packaging for the ROI calibration plate You can use the plate to calibrate a ViiA 7 System 3 times for up to 6 months if it is stored in its sleeve 3 Vortex and centrifuge the plate a Vortex the
201. is included with the spectral calibration kit The plates array cards supplied in the kit contain a buffer that accurately simulates the reagents used for PCR and therefore produces high quality calibration data If a background plate or array card froma spectral calibration kit is not available you can create one as described below Applied Biosystems Optical 96 384 Well Reaction Plate Safety glasses Optical Adhesive Cover or Optical Flat Caps Pipettor 200 uL with pipette tips Powder free gloves Deionized water Applied Biosystems Array Cards Applied Biosystems Array Card Staker Sealer Centrifuge with array card buckets and array card carrier clips Permanent marker or pen Pipettor 200 uL with pipette tips Powder free gloves Safety glasses Deionized water Create a background plate 166 IMPORTANT Wear powder free gloves while creating the background plate Remove an Applied Biosystems 96 384 Well Optical Reaction Plate from its box and place it on a clean dry surface Aliquot 20 uL deionized water to each well of the reaction plate Seal the plate using an optical adhesive cover or optical flat caps Use the plate for background calibration as you would a background plate from the spectral calibration kit Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix C Creating Custom Calibration Plates and Array Cards C Create a bac
202. it The fluorescent dyes in the array card are photosensitive Prolonged exposure to light can diminish the fluorescence of the dye Run the experiment 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a ViiA 7 Instrument to your list before you can manage it 3 After the ViiA 7 Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the RNase P wizard a Click Maintenance then click RNase P Run b In the RNase P Run screen click Start RNase P Run 5 Complete the calibration as instructed by the wizard When the side door opens load the RNase P plate or array card Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the bar code facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained Applied Biosystems ViiA 7 Real Time PCR System User Guide 79 Calibration Maintenance Networking and Security 80 Chapter 2 Calibration and Maintenance Verify the instrument performance 6 After loadi
203. ith the waste material and meet federal state and local requirements for container storage e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS e Handle chemical wastes in a fume hood e After emptying a waste container seal it with the cap provided e Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory e Ensure the health and safety of all personnel in your laboratory e Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Applied Biosystems ViiA 7 Real Time PCR System User Guide 233 Ca
204. kground plate or array card Create a background array card 1 Remove an Applied Biosystems Array Card from its box and place it on a clean dry surface 2 Using a permanent marker write Background on the side of the empty card 3 Pipette 100 uL of deionized water into each of the eight reservoirs in the card a Place the array card on a lab bench with the foil side down b Load 100 uL of the solution into a pipette c Hold the pipette in an angled position 45 degrees and place the tip into the fill port There is a fill port on the left arm of each fill reservoir the larger of the two holes Fil Z port 1 2 3 4 5 6 O O CE d Dispense the fluid so that it sweeps in and around the fill reservoir toward the vent port When pipetting the reagents into the array card pipette the entire 100 uL volume into the fill reservoir but do not go past the first stop of pipettor plunger or you may blow the solution out of the port IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port 4 Centrifuge and seal the array card as explained in Fill the array cards on page 38 Applied Biosystems ViiA 7 Real Time PCR System User Guide 167 Calibration Maintenance Networking and Security C Appendix C Creating Custom Calibration Plates and Array Cards Create a custom dye plate for calibration Create a custom dye p
205. l Calibration Plate with ROX Dye 4432402 Fast 96 Well Spectral Calibration Plate with SYBR Green Dye 4432408 Fast 96 Well Spectral Calibration Plate with TAMRA Dye 4432414 Fast 96 Well Spectral Calibration Plate with NED Dye 4432420 Fast 96 Well Region of Interest ROI and Background Plates 4432426 e Fast 96 Well Region of Interest ROI Calibration Plate e Fast 96 Well Background Plate Fast 96 Well Normalization Plates with FAM ROX and VIC 4432432 ROX Dyes e Fast 96 Well Normalization Plate with FAM ROX Dye e Fast 96 Well Normalization Plate with VIC ROX Dye Kit TaqMan RNase P Fast 96 Well Instrument Verification Plate 4351979 e TaqMan RNase P Fast 96 Well Instrument Verification Plate Applied Biosystems ViiA 7 Real Time PCR System User Guide 179 Calibration Maintenance Networking and Security Appendix D Parts and Materials Calibration and verification kits Array card sample block kits 180 ViiA 7 System consumable Part number Storage C Kit Array Card Spectral Calibration Dye TaqMan Array Calibration with FAM Dye TaqMan Array Calibration with VIC Dye TaqMan Array Calibration with ROX Dye TaqMan Array Calibration with ROI Dye TaqMan Array Calibration with FAM ROX Dye TaqMan Array Calibration with VIC ROX Dye TaqMan Array Background Buffer 4432376 Kit TaqMan RNase P Array Card Instrument Verification Reagents Port
206. l s identified in step 1 and step 2 are e Identical The sample block is contaminated Decontaminate the sample block see Decontaminate the sample block on page 124 e Reversed The ROI plate or array card is contaminated Discard the plate or array card then perform the uniformity calibration using a new ROI plate or array card 4 If the calibration fails again contact Applied Biosystems technical support Instrument does not eject the ROI plate The adhesive cover may have adhered the plate to the heated cover within the instrument 1 Power off the ViiA 7 Instrument 2 Wait for 15 minutes then power on the ViiA 7 Instrument and eject the plate 3 If the plate does not eject power off and unplug the ViiA 7 Instrument then open the access door 4 Wearing powder free gloves reach into the ViiA 7 Instrument and remove the plate from the heated cover then close the access door 5 Perform a background calibration to confirm that the sample block has not been contaminated Instrument malfunction Multiple possible causes Contact a local Applied Biosystems Field Service Office Applied Biosystems ViiA 7 Real Time PCR System User Guide 59 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Perform the dye calibration Perform the dye calibration Materials required 96 Well Plate Sample Block 384 Well Plate Samp
207. lag setting Droplets on the sides of the wells Improper sealing or seal leaks Condensation on the reaction plate Inconsistent volumes across the plate Well displays the NOAMP flag indicating the sample did not amplify Missing template Excitation source in the instrument stopped functioning Well displays the NOISE flag indicating the well produced more noise in the amplification plot than other wells on the plate Droplets on the sides of the wells Improper sealing or seal leaks Condensation on the reaction plate If a well is flagged confirm the results 1 Select the flagged well s and the associated replication group s in the plate layout or well table 2 View the amplification plot R vs Cycle and review the data for abnormalities Hold the plate or array card up to a light source and check for condensation or evaporation If there are differences in fluid volumes check the heat seal of the wells with lower volumes for signs of damage or evaporation Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit Repeat the experiment and make sure to properly seal and centrifuge the RNase P plate If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance 84 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter
208. late for calibration The Applied Biosystems ViiA 7 Real Time PCR System can be used to run assays designed with custom dyes dyes not manufactured by Applied Biosystems Custom dyes must excite between 455 and 672 nm and read between 505 and 723 nm Before you use custom dyes Before using custom dyes with the ViiA 7 System you must e Determine optimum dye concentration e Create a custom dye plate e Add the custom dye to the software e Perform a dye calibration Materials required e Centrifuge with plate adapter e Custom dye s e Safety glasses e Powder free gloves e MicroAmp Optical 96 384 Well Reaction Plate e Optical Adhesive Cover e Pipettors and pipette tips 200 uL and 1000 uL e Tubes 2 mL and 10 mL e Deionized water Determine optimum dye concentration Note Wear powder free gloves while creating the dye plate 1 Prepare and load the custom dye plate a In the center of a 96 384 well plate prepare a dilution series of the custom dye for example 25 50 100 200 400 800 1600 and 3200 nM using 20 uL volumes for a 96 384 well plate b Seal the reaction plate using an optical adhesive cover c Load the prepared reaction plate 168 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix C Creating Custom Calibration Plates and Array Cards C Create a custom dye plate for calibration 2 Start the calibration wizard a
209. late for the attributes above then the assay information file values always take precedence e Template file edt e Plate setup file txt All values in the template file take precedence except for e Target Assay SNP to Well Assignment e Biological Group Color e Sample to Well Assignment e Target Color e Task to Well Assignment e Gene Expression Targets Definition e Biological Group to Well Assignment e Genotyping Assay Definition e Well Quantity to Well Assignment e Passive Reference e Sample Color e Template file edt e Plate setup file txt e Assay information file aif aix All values in the template take precedence except for the following The following assay information file values take precedence over Plate Setup and Template e Gene Expression Targets Detectors Definition e GT Assay Marker Definition e Passive Reference The following Plate Setup values take precedence over the template e Block Type e Well Quantity to Well Assignment e Target Assay Marker to Well Assignment e Sample Color e Sample to Well Assignment e Biological Group Color e Task to Well Assignment e Target Color e Biological Group to Well Assignment 186 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference E ViiA 7 Software command line application Running the command line application from a command pr
210. le Exports data for all experiments into one contiguous data file e multiple Exports data for each experiment to a separate data file Example s multiple x lt filepath gt Required Specifies the file format of the exported file e VIIA 7 export format txt xls or xlsx e SDS23 export format txt e RDML export format rdml Example x xdml Applied Biosystems ViiA 7 Real Time PCR System User Guide 189 Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference ViiA 7 Software command line application Examples Batch file creation Results export 190 The following example uses all parameters described in Command syntax and arguments on page 188 required and optional to generate a set of experiment files cmdlineutil exe expgen t D Applied Biosystems ViiA7 Software vl 1 experiments templates standard_curve edt a D Applied Biosystems ViiA7 Software vl 1 experiments examples AIF AIF_820629 txt s D Applied Biosystems ViiA7 Software vl 1 experiments examples Plate Setup Files SDS_820629 txt m D Applied Biosystems ViiA7 Software vl 1 experiments examples SampleNames SampleFileNames txt c alloptionsused f Plate Barcode_Custom Name Field b C ViiA7 barcodes v12 txt 1 C ViiA7 Experiment For this example the command line application Imports assay definitions from the AIF_820629 txt assay information file Imports
211. le Block 60 During a dye calibration the Applied Biosystems ViiA 7 Real Time PCR System e Collects spectral data from a series of dye standards e Stores the spectral information for the dye standards in a dye calibration file The ViiA 7 Software uses the pure spectra data during experiment runs to characterize and distinguish the individual contribution of each dye in the total fluorescence collected by the ViiA 7 Instrument After each run the ViiA 7 Software receives data in the form of a raw spectra signal for each reading It determines the contribution of each fluorescent dye used in the sample by comparing the raw spectra to the pure spectra calibration data When you save an experiment TM after analysis the ViiA 7 Software stores the pure spectra with the collected fluorescence data for that experiment IMPORTANT You must calibrate only those dyes that are present in the chemistries that you intend to run on your ViiA 7 System e 96 Well Spectral Calibration Plates 96 Well Spectral Calibration Plate with FAM Dye 96 Well Spectral Calibration Plate with VIC Dye 96 Well Spectral Calibration Plate with ROX Dye 96 Well Spectral Calibration Plate with NED Dye 96 Well Spectral Calibration Plate with TAMRA Dye 96 Well Spectral Calibration Plate with SYBR Green Dye e Centrifuge with plate adapter e Powder free gloves e Safety goggles e 384 Well Spectral Calibratio
212. le block and cause an abnormally high background signal Confirm that the centrifuge you use is clean Before centrifugation wipe down the bucket using a tissue Plates only Vortex all calibration plates to ensure complete mixing then centrifuge them to ensure that all reagents are contained in the bottom of the wells The calibration plates must be well mixed and centrifuged before use Plates only Do not discard the packaging for the calibration plates Each plate can be used to calibrate the ViiA 7 System 3 times for up to 6 months if it is stored in its packing sleeve Plates only Handle the calibration plates with care to prevent contamination Do not place the plates on a lab bench to avoid contaminating them Always put calibration plates back into their packaging sleeves 96 well plates only If you are using cap strips to seal your plates firmly seal all wells before running the plate Partially seated caps can leak during the experiment causing evaporation Tubes only Firmly seal all individual tubes and tube strips Partially seated caps can leak during the experiment causing evaporation Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform regular data maintenance Perform regular data maintenance Maintain the computer hard drives Defragment and clean up the hard drive e Atleast once every month e
213. lect is unusable Analysis status Action Passed Click Next then remove the plate or array card when the ViiA 7 Instrument ejects the tray arm Failed Troubleshoot the failed calibration as described in Troubleshoot the ROI calibration on page 47 WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can be heated to 100 C Before removing the plate wait until it reaches room temperature IMPORTANT If the ViiA 7 Instrument does not eject the plate remove the plate as explained in Troubleshoot the ROI calibration on page 47 9 Discard or store the plate or array card Consumable Action Array card Discard the array card if you do not plan to perform a uniformity calibration soon Note You can reuse the array card if the ROI and uniformity calibrations are performed on the same day Plate Return the ROI calibration plate to its packaging sleeve If you plan to perform background and uniformity calibrations e During the next 8 hours keep the ROI calibration plate at room temperature The ROI calibration plate is used in the uniformity calibration e Another day return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate is stored in its original packaging sleeve you can use it to calibrate a ViiA 7 System 3 times for up to 6 months after you open it 10 In the ROI Calibration
214. libration Maintenance Networking and Security F Appendix F Safety Chemical safety Biological hazard safety General biohazard 234 AN WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories http www cdc gov biosafety publications index htm e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials e Additional information about biohazard guidelines is available a
215. ltage Power fluctuations can adversely affect the function of the ViiA 7 System Note A power line regulator monitors the input current and adjusts the power supplied to the ViiA 7 System or computer It does not protect against a power surge or failure Uninterruptible We recommend the use of a 1 5 kVA uninterruptible power supply UPS especially in power supply UPS areas prone to power failure Power failures and other events that abruptly terminate the function of the ViiA 7 System can corrupt data and possibly damage the computer or the instrument IMPORTANT UPSs provide power for a limited time They are meant to delay the effects of a power outage not to serve as replacement power sources In the event of a power loss power off the instrument and the computer unless you expect to regain power within the battery life of the UPS Surge protector We recommend the use of a 10 kVA surge protector line conditioner in areas with frequent electrical storms or near devices that are electrically noisy such as refrigerators air conditioners or centrifuges Short duration high voltage power fluctuations can abruptly terminate the function of and thereby damage the components of the computer and the ViiA 7 Instrument Note A dedicated line and ground between the ViiA 7 System computer and the building s main electrical service can also prevent problems caused by power fluctuations Applied Biosystems ViiA 7
216. mart Monitoring Service Product Bulletin Leveraging the power of the Internet while maintaining system security PN 121PBO07 03 Set the Internet Protocol TCP IP Properties for either DHCP or Static IP communication Network A Action service DHCP Touch Obtain an IP address automatically then touch Save Static IP 1 Touch Use the following IP address 2 Touch the IP Address field enter the IP address using the keypad then touch Done 3 Repeat step 2 to assign the e P addresses for the DNS Servers primary and secondary e Subnet Mask setting e Default Gateway setting 4 Touch Save to save the settings then touch OK when prompted 6 Touch to return to the Main Menu 154 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix A Manual Instrument Operation A Administrate the instrument from the touchscreen Define the system shortcuts You can use the ViiA 7 Instrument touchscreen to map the shortcut buttons that appear in the Main Menu You can configure shortcuts to automatically open specific files and folders so that you can access data quickly and easily without having to navigate to it Define the shortcuts 1 If the instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press OF 2 In the Main Menu access shortcut settings Touch Edit or e Touch Settings then touch Set Up Shortcuts
217. mats 2 0 000 eee eee eens 191 CONVENTIONS asean 24 ohh nee Rane Se ae aed oe ee tose Oe hee Se aang 191 Plate setup file format n nuanua nann nanan rereana 192 Sarmple file format sr terii ot aar aa eee ea eee ei ees 197 Baficode filestormat reri sen sips sae edie she eee wa dns Hes ne Ps ee 198 Assay information file ananunua annann Shane heed oot eee hong cee id 198 Export formats and file specifications 2 2 cece cece eens 199 Export formats eere nna ee nies ee oP Ais pees Soe cee See 199 ViA export format sssi oie ceeds enidan te toda weds e cab Aa a E 200 7900 export for Matses raa eid hehe na debe ted owed be Ged Pen Seep E ences 216 RDMicexporttormat reis v4 goer ad Botte tetas eee oes betel Bites 222 Applied Biosystems ViiA 7 Real Time PCR System User Guide 11 Calibration Maintenance Networking and Security Contents 12 APPENDIX F Safety mearen Sec aeetindia bade tend eadeencieeouwnceneenee ies 223 Instrumentation safety 2 0 20 cece eee eee eee eeee 224 Symbols on instruments 02 000 e eee eee eee eee eee 224 Locations of safety labels on instruments 2220ce cece eee eens 226 General instrument safety 0 00 cece cece cece eee eee eee eees 227 Physical hazard safety lt ictikccrc dette vets wae eta wees aad Wiens oe 228 Electrical satety e226 cee ete re ake ane sie te ene eat eee ate See 228 Bar code scanner laser safety 0 0
218. me PCR System User Guide 107 Calibration Maintenance Networking and Security Permissions and default user roles Chapter 4 Security Audit and Electronic Signature Manage user accounts default user accounts The following table shows all user configurable permissions and the settings for the Permissions Default user roles Category Function Scientist Technician Administrator Setup Create and edit experiments or experiment Yes Yes Yes templates includes running experiments Run Perform a run using the Quickstart function Yes Yes Yes Start a run Yes Yes Yes Stop a run Yes Yes Yes Targets Create targets Yes Yes Yes Library Edit targets Yes Yes Yes Delete targets Yes No Yes Analysis Create analysis settings includes default settings Yes Yes Yes lca Edit analysis settings includes default settings Yes Yes Yes Delete analysis settings Yes No Yes Run Methods Create a run method Yes Yes Yes Library Delete a run method Yes No Yes Dye Library Create a custom dye Yes Yes Yes Delete a dye Yes No Yes Study Create or edit a study for example edit a plate or Yes Yes Yes analysis settings Add transfer experiments to a study Yes Yes Yes Remove experiments from study Yes Yes Yes Preferences Edit the system preferences Yes No Yes Export the system preferences No No Yes Import the system preferences No No Yes Calibrations Perform calibrations Yes Yes
219. mical safety 0 6 scabs pone sawed bihaw sds si EATA 231 SDSS aa E ote RT ne en a ne ea E eo ee 232 Chemical waste safety 6 ccc eens 232 Biological hazard safety 0 6 ccc eee een eens 234 E Safety Alerts sccss hae see ceeded ene aed kani aie aiaia stent ahh we sehen ea ES 235 General alerts for all chemicals 00 235 General alerts for instrumentation 0000s 235 Specific alerts for instrumentation 0 eee eee eee 235 Applied Biosystems ViiA 7 Real Time PCR System User Guide 223 Calibration Maintenance Networking and Security Appendix F Safety Instrumentation safety Instrumentation safety Symbols on instruments The following table describes the electrical symbols that may be displayed on Applied Biosystems instruments Description Symbol Description Indicates the On position of the main power switch Indicates the Off position of the main power switch Indicates a protective grounding terminal that must be connected to earth ground before any other electrical connections are made to the instrument Indicates a terminal that may be connected to the signal ground reference of another instrument This is not a protected ground terminal Indicates a terminal that can ED receive or supply alternating current or voltage Indicates that the device ooo receives or supplies direct current or voltage The follo
220. mple block in the interior of the ViiA 7 Instrument 24 Applied Biosystems ViiA 7 Real Time PCR System User Guide The ViiA 7 System consists of the components shown in the following figures Calibration Maintenance Networking and Security Chapter 1 Getting Started 1 ViiA 7 System hardware Rear view Ethernet Ethernet USB USB 1 2 1 2 Component Description A Ethernet 1 An RJ45 port that provides Ethernet Gigabit communication with the port ViiA 7 Instrument IMPORTANT The Ethernet 2 port is for Applied Biosystems use only B USB ports Provide USB communication with the ViiA 7 Instrument They can be used to transfer data to from the instrument and to update the firmware Note If multiple USB drives are plugged into the ViiA 7 Instrument the instrument mounts only the first drive that is installed regardless of the USB port used C RS232 port Provides serial communication between the ViiA 7 Instrument and the computer IMPORTANT The serial port is reserved for Applied Biosystems use only D Instrument Cool the interior of the ViiA 7 Instrument fens IMPORTANT The fans must be unobstructed to ensure adequate cooling and proper function of the ViiA 7 Instrument E On Off switch Power switch for the ViiA 7 Instrument where the states are on or off
221. must add a ViiA 7 Instrument to your list before you can manage it 3 After the ViiA 7 Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenance then click Background b In the Background Calibration screen click Start Calibration 5 Click Next then perform the calibration as instructed When the side door opens load the background plate or array card Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the bar code facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained 6 After loading the plate or array card start the calibration a In the Setup tab select Check the box when the background calibration plate has been loaded then click Next b In the Run screen click START RUN IMPORTANT Do not attempt to open the access door during the run The door is locked while the ViiA 7 Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature Applied Biosystems ViiA 7 Real Time PCR System User Guide 51 Calibration Maintenance
222. n backlight and enters low power mode Define the heated cover temperature setting a b C Select Cover Idle Temperature to activate the feature Touch the Cover Idle Temperature field Enter the temperature 50 to 110 C that the heated cover should maintain when the ViiA 7 Instrument is idle then touch Done Touch Save to save the settings then touch OK when prompted Touch to return to the Main Menu Define the maintenance reminders You can use the ViiA 7 Instrument touchscreen screen to Set the reminders 1 Set the expiration period for the instrument calibrations and lamp replacement Activate deactivate or change the frequency of the maintenance reminders displayed by the ViiA 7 Instrument If the instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press In the Main Menu touch Settings then touch Set Maintenance Reminders Configure the maintenance reminders For each maintenance reminder a Touch the Calibration expires after field enter the number of days or hours that should elapse until the association calibration expires then touch Done Touch the check box to activate or deactivate reminders for the associated calibration Touch the Display reminders before field enter the number of days before the associated calibration expires that the Vii A 7 Instrument should start displaying warnings of the impending expiration then touch Done
223. n Plates 384 Well Spectral Calibration Plate with FAM Dye 384 Well Spectral Calibration Plate with VIC Dye 384 Well Spectral Calibration Plate with ROX Dye 384 Well Spectral Calibration Plate with NED Dye 384 Well Spectral Calibration Plate with TAMRA Dye 384 Well Spectral Calibration Plate with SYBR Green Dye e Centrifuge with plate adapter e Powder free gloves e Safety goggles Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the dye calibration Array Card Sample e Applied Biosystems Array Cards filled with Block FAM Dye VIC Dye ROX Dye e Applied Biosystems Array Card Staker Sealer e Centrifuge with centrifuge array card carrier clips and buckets e Powder free gloves e Safety goggles e Pipettor 200 uL with pipette tips When to perform the dye calibrations Perform a dye calibration every 6 months or as often as necessary depending on instrument use IMPORTANT You must calibrate only dyes that are present in the chemistries that you intend to run on the ViiA 7 System For example if you intend to run a TaqMan RNase P plate or array card to verify instrument performance see page 74 you must calibrate the FAM dye TAMRA dye and ROX dye because all three are present in the TaqMar assay chemistry present on the consumable IMPORT
224. n plate must be well mixed and centrifuged c Verify that the liquid is at the bottom of each well of the reaction plate If not centrifuge the reaction plate again at a greater rpm and for a longer time IMPORTANT Do not allow the bottom of the RNase P plate to become dirty Fluids and other contaminants that adhere to the bottom of the reaction plate can contaminate the sample block and cause an abnormally high background signal Correct Incorrect Ps Liquid is at e Not centrifuged with enough force or bottom of well e Not centrifuged for enough time Applied Biosystems ViiA 7 Real Time PCR System User Guide 77 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Verify the instrument performance Prepare the array IMPORTANT Wear powder free gloves while preparing the array cards card for the instrument noua 1 Remove the Array Card RNase P Kit from the freezer then allow it to thaw at verification run room temperature 2 Remove an array card from its box and place it on a clean dry surface 3 Using a permanent marker mark the side of the empty array card with RNase P 4 Transfer 100 uL of each solution into the appropriate port of the array card 11 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2
225. n to disable and enable security control restrictions and security policies for all user accounts and set up notifications when certain security events occur IMPORTANT If you disable security you inactivate audit and electronic signature functions however no audit record is generated to indicate that audit and electronic signature functions are disabled Note Security is enabled by default To enable or disable security 1 In the ViiA 7 Software select Tools Security gt Settings 2 In the Security Settings dialog box select the System tab 3 Select or deselect Enable Security Note the following e Disabling Security inactivates Auditing and E Signature e The enable commands are grayed when a run is in process e When security is disabled the 2 is not active in lower parts of the screen e The software requires you to enter your user name and password when you enable security IMPORTANT If you enable or disable the ViiA 7 Software security auditing and electronic signature feature you must similarly enable or disable the ViiA 7 Instrument security see page 156 The ViiA 7 Software cannot connect to ViiA 7 Instruments that do not match security settings 4 Click Apply Settings Applied Biosystems ViiA 7 Real Time PCR System User Guide 103 Calibration Maintenance Networking and Security 4 Chapter 4 Security Audit and Electronic Signature Configure the security system Set
226. nagement Settings Update Update function management Function Access Manager Update Update function access management Function Create Create function Delete Delete function Update Update function Role Permissions Create Create a user rolet Delete Delete a user rolet Update Modify user role permissions Audit Reason for Change Delete Create reason for change Update Delete or modify reason for change Event Manager Update Update the event manager E signature Manager Update Enable or disable e signature E signature Type Create Create an e signature meaning Delete Delete an e signature meaning Update Edit an e signature meaning or an e signature action E signature Function Update Edit an action requiring e signature Creates one role assignment record for each permission in a role 112 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators 4 Generate audit reports Review the action The Action Record log lists system specified audit events log All items in the action log are audited silently except for the items noted as configurable Configurable items may include comments in the action log View and print audit 1 reports 6 Audit Settings Update Auditing Event Archive Restore Purge Configuration Import Export Data Audit Archive Restore
227. nce information for the ViiA 7 Instrument and describes how to prepare maintain and troubleshoot the system Audience This user guide is written for laboratory staff who operate and maintain the ViiA 7 System Assumptions This guide assumes that your ViiA 7 System has been installed by an Applied Biosystems service representative This guide also assumes that you have e Familiarity with Microsoft Windows operating system e Knowledge of techniques for handling and preparing DNA samples for PCR e A general understanding of data storage file transfers and copying and pasting Applied Biosystems ViiA 7 Real Time PCR System User Guide 13 Calibration Maintenance Networking and Security About This Guide Safety information Safety information Note For general safety information see this section and Appendix F Safety on page 223 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit u
228. ned by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners TaqMan is a registered trademark of Roche Molecular Systems Inc Twister is a trademark of Caliper Life Sciences Apple Safari and Macintosh are trademarks of Apple Inc Microsoft and Internet Explorer are trademarks of Microsoft Corporation Mozilla is a trademark of Mozilla Foundation 2010 Life Technologies Corporation All rights reserved Part Number 4442661 Rev C 12 2010 Contents About This Guide oc e cc sceaticovcien deci denteciesvinaciciad 13 PU DOSE anes ienne bard ety ee AP een ee ead eet Gann SOR eae EA 13 OTe CL sina cect cata EEE Davee aap E EAE aoe E I E E T space hanes 13 ASSUMPIONS iie eee are eer E ee ee 13 Safety information 6 00 ccc ee teen anaran 14 Safety alert Words reie ie ade peace pene a Ee ELLER YEAS O Ped bee 14 DSS eee eect aes teeter eee eee eects ee ee ee ec eee 14 Safety labels on instruments 0 000 cece cece eee eeeees 15 CHAPTER 1 Getting Started 2 ccccccccetscnitecetecenaccsaseseresosucane 17 About the ViiA 7 System cnn nnn n een enn e nee eneenes 18 About data collection v 00 0i4 sence tte eeeiadane T E EErEE EAR EEEE EEES 18 Instrument filters and supported dyes 2 20c cece eee eens 19 Specifications and layo
229. negative control NC no template control NTC nonfluorescent quencher minor groove binder NFQ MGB normalization calibration normalized quantity Applied Biosystems ViiA 7 Real Time PCR System User Guide Glossary A software feature that allows you to view information about instruments on the network In the Instrument Console you can monitor the status of any instrument on the network view calibration maintenance and instrument properties for a selected instrument and open and close the instrument drawer A software feature that allows you to view information about instrument available on the network In the Instrument Manager you can monitor the status of an instrument monitor amplification plots and temperature plots in real time view the calibration status perform calibrations and manage files on the instrument including downloading completed experiments to your computer In presence absence experiments a short synthetic DNA template that is added to PCR reactions The IPC can be used to distinguish between true negative results the target is absent in the samples and negative results caused by PCR inhibitors incorrect assay setup or reagent or instrument failure See internal positive control IPC See negative control IPC wells In the thermal profile a stage with a temperature increment to generate a melt curve The temperature at which 50 of the DNA is double stranded and 50 of the DNA is diss
230. nfiguration protocol DHCP operation are connected to anetwork by their Ethernet 1 ports In this layout any computer on the network can monitor or control the ViiA 7 Instrument Experiments can be started remotely from the networked computer or locally from the ViiA 7 Instrument touchscreen Note A networked computer running the ViiA 7 Software can simultaneously control up to 4 instruments and monitor up to 15 instruments that have been connected to the network DHCP server Networked Networked computer instrument s The ViiA 7 System computer can be connected to the network In the configuration shown below computers on the network can exchange experiment data with the ViiA 7 System computer however the ViiA 7 Instrument can be neither monitored nor controlled remotely because it is physically isolated from the network DHCP server Networked Networked Instrument computer computer Applied Biosystems ViiA 7 Real Time PCR System User Guide 89 Calibration Maintenance Networking and Security 3 Chapter 3 Networking Network setup workflow Networking guidelines and best practices Consult a network administrator We recommend that you consult a network administrator before connecting the ViiA 7 System to your laboratory network To enable the full functionality of the ViiA 7 Software the computer requires a network connection Limit remote m
231. ng the plate or array card start the calibration a In the Overview screen select Check the box when the RNase P calibration plate has been loaded then click Next b In the Run screen click START RUN to start the calibration IMPORTANT Do not attempt to open the access door during the run The door is locked while the ViiA 7 Instrument is in operation Note Before starting the calibration the instrument may pause up to 10 minutes to allow the heated cover to reach temperature When the run is complete and the ViiA 7 Software displays the Analysis screen verify the status of the run e Passed The ViiA 7 System passed the RNase P run Go to step 12 on page 82 e Failed The ViiA 7 System failed the RNase P run Go to step 8 to review the data for outliers If the run fails the ViiA 7 Software may have included outliers that caused the initial analysis to fail Experimental error may cause some wells to be amplified insufficiently or not at all These wells typically produce Cy values that differ significantly from the average for the associated replicate wells If included in the calculations these outlying data outliers can result in erroneous measurements Inthe Amplification Plot select Ct vs Well from the Plot Type menu then verify the uniformity of each replicate population on the reaction plate controls standards and unknowns by comparing the groupings of Cy values a In the
232. ning 122 EMC standards 230 enable calibration reminders 96 security instrument 156 security software 103 ergonomics safety 229 error 243 e sig See electronic signature Ethernet 1 port 25 88 91 define IP settings 154 experiment document 243 experiment name 244 experiment type 18 244 experiments archive 37 create from touchscreen 143 RNase P instrument verification 74 run from touchscreen 144 transfer to from the instrument 95 145 export 244 7900 file format 216 audit records 114 audit settings 120 e sig settings 120 RDML file format 222 security settings 120 user account settings 120 ViiA 7 file format 200 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Index export formats 199 7900 file 216 RDML file 222 ViiA 7 file 200 F FAM dye 19 62 fans instrument 25 feet 24 27 file assay information 185 198 barcode 185 198 export formats 199 import formats 191 plate setup 192 sample 185 197 setup 185 fill array cards calibration 38 instrument verification 78 filter 244 filter sets 19 firmware update 132 150 flag 244 fluorescence background 49 format 7900 export file 216 assay information file 198 barcode file 198 plate setup file 192 RDML export file 222 sample file 197 ViiA 7 export file 200 forward primer 244 fuse cover 25 28 fuse replacement 130 G guidelines chemical safety 231 chemical waste disposal 232 chemical wa
233. nknown y intercept 252 e In amplification plots the level of fluorescence above the baseline and within the exponential growth region For the Baseline Threshold algorithm the threshold can be determined automatically or can be set manually e In presence absence experiments the level of fluorescence above which the software assigns a presence call The PCR cycle number at which the fluorescence meets the threshold in the amplification plot Instrument display that you touch to control the instrument Type of calibration in which the software measures sample block uniformity The calibration generates data that compensate for the physical effects of the ViiA 7 System filters on data collected during an experiment In the software the task for the target or SNP assay in wells that contain the sample being tested In quantification experiments the task for the target in wells that contain a sample with unknown target quantities In genotyping experiments the task for the SNP assay in wells that contain a sample with an unknown genotype In presence absence experiments the task for the target in wells that contain a sample in which the presence of the target is not known In melt curve experiments the task for the target in wells that contain a sample with an unknown melt curve profile In the standard curve the value of y where the regression line crosses the y axis The y intercept indicates the expected threshold cycle Cy
234. ns the label that defines the beginning of the sample setup data Example Sample Setup Sample setup column header The column header contains the headings that define the positions of the data columns in the sample setup body The headings are separated by tab characters See Plate setup data columns on page 194 for a list of the data column headers Example Well Sample Name Sample Color Biogroup Name Biogroup Color Target Name Sample setup body Contains the sample setup data where each row defines the contents of a single well on the consumable including the well contents sample target or SNP assay added to the well task assignments and comments If a well contains multiple assays multiplex PCR the data for the additional assays are defined on separate lines by repeating the well designation See Plate setup data columns on page 194 for a list of the data column headers Note The sample setup data rows can occur in any order Example Well Sample Name Sample Color Biogroup Name Biogroup Color Target Name 1 Liver cDNA RGB 25 0 0 2 Liver cDNA RGB 25 0 0 3 Liver cDNA RGB 25 0 0 4 Heart cDNA RGB 0 25 0 5 Heart cDNA RGB 0 25 0 Applied Biosystems ViiA 7 Real Time PCR System User Guide 193 Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference Import formats and file specifications Plate setup data columns The following table lists t
235. nsists of the components shown in the following figure Component Description ViiA 7 Instrument Performs fluorescence research detection and data collection of experiment and calibration consumables Computer C Monitor Keyboard L Mouse Run the ViiA 7 Software that is used to e Calibrate the ViiA 7 Instrument e Set up experiments e Optional Run experiments e Analyze experiments Bar code reader Scans the bar codes of consumables before and after they are loaded into the ViiA 7 Instrument Twister II Robot Automates loading and unloading of consumables to and from the ViiA 7 Instrument Connection Description A Power cables Supply power to the computer the Applied Biosystems Twister II Robot and the ViiA 7 Instrument 8 B LAN connection or Ethernet cablet Connects the ViiA 7 Instrument Ethernet 1 port to the Ethernet port on the network interface card in the computer DVI cable Connects the monitor to the computer DVI port Connects the bar code reader to the computer USB port Keyboard cable Connects the keyboard to the computer USB port C D Bar code reader cable E F Mouse cable Connects the mouse to the computer USB port G Serial cable Connects the Twister II Robot to the computer serial port
236. nstrument settings 37 148 normalization calibration 69 reminders 153 RNase P instrument verification experiment 74 ROI calibration 42 schedule 34 software licenses 133 uniformity calibration 55 materials accessories 176 consumables 181 kits 177 mDNS support 88 melting temperature Tm 245 monitoring instrument over a network 88 monthly maintenance tasks 34 moving and lifting safety computers and monitors 227 instrument 227 moving parts safety 228 moving the instrument 163 MSDSs about 14 description 232 obtaining 232 240 N name instrument 152 NED dye 19 62 negative control NC 245 network computer setup 92 guidelines 90 instrument setup 91 layouts 89 overview 88 settings instrument 154 no template control 245 nonfluorescent quencher minor groove binder 245 257 Index normalization calibration 69 245 data 69 perform 71 troubleshoot 73 when to perform 69 normalized quantity 245 normalized quantity mean 246 normalized quantity SE 246 normalized reporter Rn 246 notifications maintenance reminders 153 security auditing electronic signature 105 NTC 245 0 object audit history display 111 114 119 omit outliers 79 omit well 246 online Help See Help system operating system update 131 optical calibration perform 57 troubleshoot 53 order calibration and verification kits 177 from the software 174 from the website 175 how to 174 outlier 246 outlier removal 79 outlier removal for in
237. nt Software Downloads page click Updates Patches The website opens the page describing the latest software and firmware updates for the ViiA 7 Software and ViiA 7 Instrument Update the ViiA 7 Software Prepare for the To update the ViiA 7 Software prepare your computer by exporting the application upgrade libraries and backing up your experiment files To prepare for the software update 1 Back up the application libraries For each library a In the main menu of the ViiA 7 Software select Tools gt lt desired library gt b When the library dialog box opens select the element s to export then click Export c In the Export dialog box click Save to archive the selected records 2 Back up all experiment files by creating a copy of the directory that you are using to store files The default directory for experiments is D Applied Biosystems ViiA7 Software 1 1 experiments Install the software Install the software update according to the instructions that download with the software If you are installing the update to a computer that already contains the ViiA 7 Software the update automatically acquires the software license from the existing installation If you are installing the ViiA 7 Software to a computer that does not contain a previous installation you must have a license file supplied by Applied Biosystems If you do not have a license file obtain one as explained in Manage ViiA
238. nt settings 000000eeeeeeeeee 148 Perform an instrument self test 2 0 0 0 cece eee eee eee eens 149 Update the instrument firmware 200000 cece eee eens 150 Applied Biosystems ViiA 7 Real Time PCR System User Guide 9 Calibration Maintenance Networking and Security Contents 10 APPENDIX B APPENDIX C Administrate the instrument from the touchscreen ccc eee cece eee eee 151 Define the date and time 00 cece een nett eee e eae 152 Define the instrument settingS c cece rnrn rnrn 152 Define the maintenance reminders 00 cece eect eee eee aee 153 Define the network settingS 0 cece cece tee eee eee 154 Define the system shortcutS 0 000 ttt e teens 155 Review the instrument statistics 0 20 0 nent raan 155 Enable or disable instrument security 0200002 cece eee eee eee 156 View the instrument log 0 cece eee eee eee ae i 157 Power On or Off Store and Move the ViiA 7 System 159 Place the ViiA 7 System on standby 0 000 c cece cece eee eee cent e ee 160 Power on the ViiA 7 System 2 0 0 ccc ce ce cnn een een nee ne eens 160 Power off the ViiA 7 System 0 cece eee e rraren 161 Store the ViA 7 Systeme fastari bon teal ee ees ek ed ee Ae ae odd 162 Materials required ss 2c 204 2 nero nd bet dk ene ata ae pe ae ae cb ry 162 Prepare the ViiA 7 Instrument
239. ntenance Networking and Security 3 Chapter 3 Networking Monitor the ViiA 7 Instrument 98 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security This chapter covers CHAPTER 4 Security Audit and Electronic Signature Section 4 1 Administrators ccc cece cece eee eee eee eee e eee e eens 101 mM Administrators Overview 0 0c ccc cette nnen errer 101 mM Configure the security system 2 eee eens 103 M Manage user accounts 0 6 cece ee teen e eee rnn 106 M Manage auditing 0 6 nent eens 110 E Generate audit reports 6 eee eee eens 111 M Manage electronic signature 0 0 6 ee eee nes 116 mM Generate e signature reports 6 1 eee eee tenes 119 m Export and import user security audit and e signature settings 120 Section 4 2 Users ios c cece cseeas copied diet aes ERKENE EEEIEE IEEE 121 M Users OVervieW 6 ec een ene rererere ereer 121 BM SCCUMY 102s cece cies a teal yas We aw ins Bible ce EE cies 121 BM Audits sac cendsice TETEE Senta eee ees E EE TE wt aes 122 M Electronic signature 6 ene 122 Applied Biosystems ViiA 7 Real Time PCR System User Guide 99 Calibration Maintenance Networking and Security Chapter 4 Security Audit and Electronic Signature 100 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security SECTIO
240. ntenance Networking and Security Appendix D Parts and Materials Accessories Accessories The following accessories are to be used with the Applied Biosystems ViiA 7 Real Time PCR System ViiA 7 System accessories Part number Applied Biosystems ViiA 7 System 384 Well Plate Adapter 4457087 Applied Biosystems ViiA 7 System 384 Well Sample Block 4453553 Applied Biosystems ViiA 7 System 384 Well Array Card Heated Cover 4453555 Applied Biosystems ViiA 7 System 96 Well Heated Cover 4453560 Applied Biosystems ViiA 7 System 96 Well Plate Adapter 4459845 Applied Biosystems ViiA 7 System 96 Well Sample Block 4453556 Applied Biosystems ViiA 7 System 96 Well Tube Adapter 4462077 Applied Biosystems ViiA 7 System Array Card Plate Adapter 4454166 Applied Biosystems ViiA 7 System Array Card Sample Block 4453554 Applied Biosystems ViiA 7 System Fast 96 Well Heated Cover 4459838 Applied Biosystems ViiA 7 System Fast 96 Well Plate Adapter 4459846 Applied Biosystems ViiA 7 System Fast 96 Well Sample Block 4453559 Applied Biosystems ViiA 7 System Fast 96 Well Tube Adapter 4462078 176 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix D Parts and Materials Calibration and verification kits Calibration and verification kits The following kits are to be used with the Applied Biosystems ViiA 7 Real
241. nts that are audited and the reasons available to users when audit mode is set to Prompt or Required Auditing is disabled by default IMPORTANT If you disable security you inactivate audit functions No audit record is generated for the inactivation of audit and electronic signature functions when you disable security 1 In the ViiA 7 Software select Tools gt Security gt Settings 2 In the Security Settings dialog box select the Audit tab 3 Select or deselect Enable Audit 4 Click Apply Settings Select objects to audit 1 Select the objects to audit and the mode for each enabled item e Experiments e Experiment Templates e Study 2 Set the Audit Mode for each item you enable for auditing e Optional The event is audited a reason prompt is displayed but the user can cancel and continue without entering a reason e Required The event is audited a reason prompt is displayed and the user must specify a reason e Silent The event is audited no reason prompt is displayed 3 Click Apply Settings Create audit reason settings You can create modify and delete the reasons that are available for selection in the Audit Reason dialog box displayed when a user performs an audited action 1 To require users to select a pre defined reason in the Audit Reason dialog box displayed when a user performs an audited action enable Require users to select a reason to change from the list Users are no
242. o right and from top to bottom The ViiA 7 Software can export the data used to generate the multicomponent plot of a real time PCR experiment The multicomponent data tracks the raw fluorescence of all reporter dyes present on the reaction consumable throughout the duration of the experiment run The section begins with a column header followed by the multicomponent data where each row contains the data for a single well separated by tab characters The multicomponent data contains a dye column for each dye present on the reaction consumable including reporter dyes quencher dyes except non fluorescent dyes and the passive reference Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Cycle The cycle of the run during which the ViiA 7 Integer Instrument recorded the fluorescence data lt Dye name gt The raw fluorescence for the designated dye Float measured by the ViiA 7 Instrument at the specified well and cycle Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems ViiA 7 Real Time PCR System User Guide 205 Calibration Maintenance Networking and Security E Appendix E ViiA 7 Software Reference Export formats and file specifications Results data 206 The ViiA 7 Software can export the results data from an analyzed experiment file The format and content of the re
243. ociated into single stranded DNA In a melt curve experiment the melt curve plot displays the melting temperature In Melt Curve experiments the region before and after the melting temperature Tm The task for targets or SNP assays in wells that contain water or buffer instead of sample No amplification of the target should occur in negative control wells Previously called no template control NTC See negative control NC Molecules that are attached to the 3 end of TaqMan probes When the probe is intact the nonfluorescent quencher NFQ prevents the reporter dye from emitting fluorescence signal Because the NFQ does not fluoresce it produces lower background signals resulting in improved precision in quantification The minor groove binder MGB increases the melting temperature Tn of the probe without increasing its length allowing for the design of shorter probes Type of calibration in which the software collects data from the normalization standards then stores it in a normalization calibration file This file is used in comparisons of data from multiple instruments within a study Either the Cy Avg of the target gene minus the Cr Avg of the endogenous control Comparative Cy experiments or the Q Avg of the target divided by the Q Avg of the endogenous control Relative Standard Curve experiments 245 Calibration Maintenance Networking and Security Glossary normalized quantity mean normalized
244. olume of 10 bleach solution into each contaminated well b Ineach contaminated well pipette the solution up and down several times to rinse the well c Pipette the bleach solution to a waste beaker Repeat steps 5 through 9 to rinse the wells of the sample block and to verify that you have eliminated the contamination IMPORTANT Always use deionized water to rinse wells after cleaning with bleach or ethanol solution If the contamination remains contact Applied Biosystems Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 5 Service 5 Replace the halogen lamp Replace the halogen lamp Replace the halogen lamp after approximately 2000 hours of life WARNING PHYSICAL INJURY HAZARD The ViiA 7 System and lamp are hot The lamp can become very hot while in use Before handling the lamp allow it to cool for 15 minutes and put on protective powder free gloves CAUTION PHYSICAL INJURY HAZARD Wear disposable powder free gloves when handling the lamp to prevent burns and to prevent shortening the life of the replacement lamp WARNING This ViiA 7 Instrument is designed for 12 V 75 W halogen lamps only Replace with halogen bulbs supplied by Applied Biosystems Materials required e Safety glasses e Powder free gloves e Halogen bulb 12 V 75 W Halogen lamp warnings The ViiA 7 Software can display the following warnings before o
245. ommended in the SDS e Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Applied Biosystems ViiA 7 Real Time PCR System User Guide 231 Calibration Maintenance Networking and Security F Appendix F Safety Chemical safety SDSs About SDSs Chemical manufacturers supply current Safety Data Sheets SDSs with shipments of hazardous chemicals to new customers They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated SDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new SDS packaged with a hazardous chemical be sure to replace the appropriate SDS in your files Obtaining The SDS for any chemical supplied by Applied Biosystems is available to you free SDSs 24 hours a day To obtain SDSs 1 Goto www appliedbiosystems com click Support then select SDS 2 In the Keyword Search field enter the chemical name product name SDS part number or other information that appears in the SDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following e Open To view the document e Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you choos
246. ompt Running the 1 In the desktop select Start Run application 2 Inthe Run dialog box enter cmd in the Open field then click OK 3 In the DOS prompt change to the installation directory and enter the command a Enter cd D applied biosystems ViiA7 then press Enter b Enter cmdlineutil exe followed by expgen or export then all applicable parameters and arguments See Command syntax and arguments on page 188 for a complete list of command line parameters Viewing the The command line application includes a help function that provides the information command line help in this chapter To view help for e The entire application enter cmdlineutil exe help e A particular function enter cmdlineutil exe expgen help to view the file generation help or cmdlineutil exe export help to view the file export help Applied Biosystems ViiA 7 Real Time PCR System User Guide 187 Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference ViiA 7 Software command line application Command syntax and arguments Batch file creation The command used to create batches of files uses the following syntax cmdlineutil ex xpgen parameters The following table lists the acceptable parameters that can be included in any order See Examples on page 190 for an example of the experiment creation command IMPORTANT Enclose file paths in double quotes to allow spaces in the string
247. on 42 Perform the ROI calibration e Every 6 months or as often as necessary depending on instrument use e After replacing the lamp IMPORTANT After every ROI calibration you must perform a background calibration uniformity calibration dye calibration normalization calibration and RNase P instrument verification experiment Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance 2 Perform the ROI calibration About the ROI calibration data During the ROI calibration the ViiA 7 Software captures images of the ROI calibration plate at each instrument filter An ROI calibration passes if the collected image for each filter shows all wells of the ROI plate or array card Each well in the image must be distinct and visible at the same luminosity relative to the other wells in the image You can review the ROI calibration image for each filter set by selecting the desired filter combination from the Filter Set menu of the ROI tab in the Instrument Manager Passing image Green circles appear around all wells indicating that the wells calibrated successfully Each green circle indicates that the region of interest for the well position is sufficiently bright nm MOM O O O CE Ok Ok Ok Ok OF OK K Jk Ok Ok OR OR OR OR OR OK i aGBeeeeeeee ee Jk J Ok Ok J OK OO Oi Jk OO Ok Ok OK OOK hE EX RO Failing image Red c
248. on technology department for the following information e Network address of a SMTP server e A username and password for the server if required for access e The Secure Sockets Layer SSL setting of the server on or off Define the mail 1 In the ViiA 7 Software select Tools gt Preferences server settings 2 In the Preferences dialog box select the SMTP Settings tab 3 In the SMTP Settings tab define the settings for the SMTP server e Outgoing Mail Server SMTP field Enter the network address of a Simple Mail Transfer Protocol SMTP server Optionally you can specify the transmission control protocol TCP port for the server by appending the port number to the server name separating the two using a colon For example smtp mycompany com 2023 Note If a TCP port is not specified the ViiA 7 Software uses the default port number 25 Encryption Required Select if the mail server has SSL enabled Authentication Required Select if the mail server requires a user name and password User Name and Password fields If the mail server requires authentication enter the user name provided by your systems administrator 4 Click OK 96 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Modify the notification settings for a monitored ViiA 7 Instrument Chapter 3 Networking 3 Monitor the ViiA 7 Instrument 1
249. on to generate experiment files validate the contents of the files by opening them in the ViiA 7 Software Command line workflows The command line interface supports the workflows in the following figure For each workflow the figure shows both the required and optional supporting files Single Experiment File Creation Workflow aera ViiA 7 Application feos Experiment Document SDS Setup Sample to Well AIF X File Barcode Template edt File txt File txt txt xmU Export Workflow c d li S il Ye lt ViiA 7 Application Experiment Document Single eds E Required 184 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference ViiA 7 Software command line application Supporting files for experiment creation The file generation function cmdlineutil ex xpgen can use the files shown below The command does not require all input files File Description assay information file aif or aix A tab delimited or XML data file that is shipped on a CD with each TaqMan assay ordered from Applied Biosystems For some products assay information files are available for download from the Applied Biosystems website following delivery The file which contains data describing the assay can be imported into the ViiA 7 Software for use in related exper
250. onent Description A On Off switch Power switch for the Twister II Robot where the states are on or off 0 Power port 100 240V port that provides power to the Twister II Robot RS232 port Provides serial communication with the computer Fuse cover Two T1 6A 250VAC 5 x 20 mm electrical fuses that protect the Twister Il Robot from excessive electrical current Rack parts and Racks are removable aluminum frames used as input and output locations for PCR functions consumables Rack positions are numbered counter clockwise with position 1 closest to the front of the Twister II Robot see below Each rack is labeled for a specific position and cannot be exchanged with the other racks Component Description A Handles For connecting or disconnecting racks from the pod Rack locator Locks the rack onto the pod in the correct position notch Note Do not drop the racks If the rack is bent the Twister II Robot cannot properly place the consumables 28 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 1 Getting Started 1 ViiA 7 System hardware Electrical protective devices We recommend several protective devices to protect the ViiA 7 System in environments with large voltage and power fluctuations Power line regulator We recommend the use of a 1 5 kVA power line regulator in areas where the supplied power fluctuates in excess of 10 of the normal vo
251. onitoring to 10 computers Avoid using more than 10 computers to simultaneously monitor the ViiA 7 Instrument remotely Although the ViiA 7 System supports remote monitoring from multiple computers each connection taxes the instrument microprocessor Too many connections can overburden the ViiA 7 System and result in instrument errors Note The effects of an overburdened ViiA 7 System are evident in the Temperature Plot during a run Symptoms can include extended hold times or brief unexpected plateaus in the instrument Temperature Plot Observe the restrictions to mDNS and Autodiscovery The ViiA 7 System supports mDNS but only when the ViiA 7 Instrument and computer share a direct network connection and are within the same subnet Consequently network computers that are separated from the ViiA 7 System by a router hub or another network device may not be able to access the ViiA 7 Instrument by its host name Confirm the uniqueness of the instrument name The ViiA 7 Instrument does support name resolution but the instrument name must be unique within the subnet The ViiA 7 Software can automatically discover ViiA 7 Instruments on the link local network that are configured for Autodiscovery see Define the network settings on page 154 Note The ViiA 7 System does not test the uniqueness of the instrument name when it is set Name ViiA 7 Instruments using lower case letters When you
252. or printed 4 Save or print the record e Click H Save select a location for the export file enter a name for the file then click Save or e Click gh Print 5 Click OK in the confirmation message Save or print the table of e signature events 1 In the ViiA 7 Software open an experiment eds or template edt file 2 In the open experiment or template click Oy Audit then click Print E Signatures 3 Save or print the record e Click Save select a location for the export file enter a name for the file then click Save or e Click gh Print 4 Click OK in the confirmation message Applied Biosystems ViiA 7 Real Time PCR System User Guide 119 Calibration Maintenance Networking and Security 4 Chapter 4 Security Audit and Electronic Signature Export and import user security audit and e signature settings Export and import user security audit and e signature settings Note The export import feature can be used to replicate identical security audit and e signature settings across multiple computers The feature allows you to create a standard security audit and e signature settings image for the ViiA 7 Software that can then be imported by other copies of the software to bypass manual setup Export 1 In any screen of the SAE module click Export 2 Select the items to export e All Contains all settings e Custom Contains select settings Users amp
253. orted from high resolution melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Sample Name The name of the sample contained by the well 100 character string Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN or NTC RQ The relative quantity calculated for the replicate Float wells of the target sample combination RQ Min The minimum relative quantity calculated for the Float replicate wells of the target sample combination The lower limit of the confidence interval RQ Max The maximum relative quantity calculated for the Float replicate wells of the target sample combination The upper limit of the confidence interval Ct Mean The average C of the replicate wells for the Float specified target sample combination Delta Ct Mean The average AC of the replicate wells for the Float specified target sample combination Delta Ct SD The standard deviation of the ACy for the replicate Float well Depending on the analysis settings this column may by replaced with Delta Ct SE the standard error of the AC Delta Delta Ct The AAC value of the replicate wells for the Float specified target sample combination Applied Biosystems ViiA 7 Real Time PCR System User Guide 209
254. plug the USB drive Applied Biosystems ViiA 7 Real Time PCR System User Guide 145 Calibration Maintenance Networking and Security A Appendix A Manual Instrument Operation Operate the instrument from the touchscreen Copy experiment 1 Plug a USB drive into the USB port on the right side of the touchscreen results to a USB drive 2 If the instrument is in standby touch the ViiA 7 Instrument touchscreen to awaken it then press Ot 3 Inthe Main Menu touch _ Collect Results 4 In the Collect Results screen touch the desired experiment touch Y Copy to USB 5 In the Copy Results To USB screen touch Y Copy to USB 6 Touch to return to the Main Menu 7 Unplug the USB drive Note After the results from a completed run have been collected the corresponding experiment displays Collected and it can be deleted 146 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix A Manual Instrument Operation Maintain the instrument from the touchscreen Maintain the instrument from the touchscreen The ViiA 7 Instrument touchscreen provides access to several maintenance functions that cannot be accessed remotely from the ViiA 7 Software The following local ViiA 7 Instrument functions are performed as part of regular ViiA 7 Instrument maintenance m Back up and restore the instrument settings 6 00 cece eee eee 148 E Perform an
255. ports 113 user report 109 protective devices electrical 29 pure dye 246 purge audit records 113 Q quantity 247 quencher 247 R R2 value 79 racks robot 28 radioactive waste handling 233 raw data plot 247 RDML 247 RDML export file format 222 reaction mix 247 reagents 247 real time PCR 247 recommended maintenance schedule 34 reference 247 reference sample 247 refSNP ID 247 region of interest ROD calibration 248 registration software 133 regression coefficients 247 248 regression line 248 regulator power line 29 reinstalling the instrument 164 reject well 248 relative standard curve method 248 reminders calibration 96 remote monitoring computer setup 92 guidelines 94 instrument 94 instrument setup 91 removal lamp 127 removal outlier 79 repetitive motion safety 229 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Index replace halogen lamp 127 instrument fuses 130 replicate group 248 replicates 248 reporter 248 reports action log 111 113 114 119 audit 113 object audit history 111 114 119 system configuration history 111 112 114 119 user 109 requirements component clearances and positioning 21 computer 30 electrical 22 environmental 22 exhaust venting 22 physical clearances 21 SMTP server 96 weight 20 restore audit records 113 instrument settings 148 results transfer to USB drive 146 reverse primer 248 reverse t
256. pport 88 moving 163 name setting 152 network setting 154 networking 88 91 normalization calibration 69 operation safety 227 power on off 160 161 RNase P experiment 74 ROI calibration 42 security 156 self test 149 settings 37 148 Smart Monitoring 154 software 30 specifications 20 standby 160 standby time out 152 static IP support 88 statistics 155 storage 162 system shortcuts 155 touchscreen 24 142 uniformity calibration 55 verification 77 78 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Instrument Console 245 Instrument Manager 245 instrument verification perform 79 troubleshoot 83 internal positive control IPC 245 IP settings Ethernet 1 port 154 IPv4 link local IPV4LL 88 is signed field 122 K keys software 133 L lamp 24 replacement 127 laser classification 229 laser safety bar code scanner 229 requirements 229 layout instrument 23 network 89 License Central 133 licenses software 133 lifespan lamp 127 line conditioner requirements 29 location requirement 22 log in user account 121 log instrument 157 logged in user name display 107 in user account 106 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Index M maintenance background calibration 48 computer hard drives 37 dye calibration 60 experiment files 37 instrument 147 i
257. proceed to the Start Run screen Applied Biosystems ViiA 7 Real Time PCR System User Guide 143 Calibration Maintenance Networking and Security A Appendix A Manual Instrument Operation Operate the instrument from the touchscreen Run an experiment 10 144 If the instrument is in standby touch the ViiA 7 Instrument touchscreen to awaken it then press Ot In the Main Menu screen then touch g When the side door opens load the appropriate plate or array card Ensure that the consumable is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the bar code facing the front of the instrument 9900000000 90000000001 In the Main Menu touch a Browse Experiments In the Experiments screen touch the desired experiment then touch either e Start Run to start the run immediately then go to step 10 or View Edit to view or edit the experiment before starting the run Modify the experiment parameters as needed To e Add a stage or step to the thermal profile touch the stage or step to the left of where you want to add the stage or step then touch afg Add e Add a melt curve to the end of the thermal profile touch Add Melt Curve e Change the time or temperature of a stage or step touch the time temperature field of the stage or step modify the settings as desired then touch Close
258. ptions then click OK 5 Click to close the Print Preview dialog box Applied Biosystems ViiA 7 Real Time PCR System User Guide 115 Calibration Maintenance Networking and Security 4 Chapter 4 Security Audit and Electronic Signature Manage electronic signature Manage electronic signature Access the e Signature Settings screen and enable or disable e sig IMPORTANT If you disable security you inactivate audit and electronic signature functions No audit record is generated for the disabling of audit and electronic signature functions when you disable security 1 In the ViiA 7 Software select Tools Security Settings 2 In the Security Settings dialog box select the e Signature tab 3 Select or deselect Enable e Signature IMPORTANT Enabling the electronic signature feature can substantially increase the size of experiment eds template edt and study edm files 4 Click Apply Settings Configure the meanings of the electronic signatures Use the Security Settings dialog box to add or remove electronic signature meanings and to determine the data types to which they apply The e signature meanings are the text that a user can select to describe a reason for an electronic signature The ViiA 7 Software is installed with the following default meanings Default data types E Signature definition Plate Thermocycler Analysis Analysis setup Protocol Protocol R
259. put Automatic Ct Whether the baseline was determined true or false Baseline automatically true or manually false Baseline Start The first cycle used to calculate the baseline Float Baseline End The last cycle used to calculate the baseline Float Efficiency The calculated efficiency of the target assay for the Float 1 to 100 specified target sample combination Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Technical analysis result study The following table describes the results data exported from high resolution melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Sample Name The name of the sample contained by the well 100 character string Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN Or NTC Omitted Indicates whether the well was omitted from the analysis true or included false true Or false RQ The relative quantity calculated for the replicate wells of the target sample combination Float RQ Min The minimum relative quantity calculated for the replicate wells of the target sample combination The lower limit of the confidence interval Float RQ
260. quantity SE normalized reporter Rn omit well outlier passive reference plate layout plate setup file point positive control primer mix primer probe mix pure dye 246 The relative standard curve equivalent of the AC mean value found in Comparative Cy experiments computed as the geometric mean The relative standard curve equivalent of the AC SE value found in Comparative Cy experiments computed as the geometric standard error of the mean Fluorescence signal from the reporter dye normalized to the fluorescence signal of the passive reference dye usually ROX dye on Applied Biosystems instruments An action that you perform before reanalysis to omit one or more wells from analysis Because no algorithms are applied to omitted wells omitted wells contain no results You can add wells back in to the analysis no information is permanently discarded A measurement such as a Cy that deviates significantly from the measurement of the other replicates for that same sample A dye that produces fluorescence signal independent of PCR amplification and that is added to each reaction at a constant concentration Because the passive reference signal should be consistent across all wells it is used to normalize the reporter dye signal to account for non PCR related fluorescence fluctuations caused by minor well to well differences in volume Normalization to the passive reference signal generally results in dat
261. r 1 to 96 384 Time The time in milliseconds after the start of the Integer run when the reading was taken Temp The temperature C of the sample when the Integer ViiA 7 Instrument recorded the fluorescence data Cycle The cycle of the run during which the ViiA 7 Integer Instrument recorded the fluorescence data lt Dye name gt The raw fluorescence for the designated dye Float measured by the ViiA 7 Instrument at the specified well and cycle Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference E Export formats and file specifications Results file When selected as an export option the ViiA 7 Software exports sample setup data to a stand alone file The sample setup file describes the sample configuration on the experiment consumable including sample and assay data positions and task assignments File header The file begins with a line that names the export format SDS 2 3 and the type of data contained by the file Std Results The following lines listed in the table below describe the qualities of the ViiA 7 Instrument and several other general experiment properties Field Description Output Filename The path to the experiment file on the local lt filename gt
262. r To ensure optimal performance of the remote monitoring feature observe the remote monitoring following guidelines e The ViiA 7 Software can monitor up to 15 instruments e We do not recommend that a ViiA 7 Instrument be monitored by more than 10 computers simultaneously e Unless you are sure that your ViiA 7 Instrument and computer exist on the same subnet we recommend that you use the IP address of the ViiA 7 Instrument to add it for remote monitoring Monitor the status of ViiA 7 Instrument during a run 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instrument from the list of instruments on the network then click Add to My Instruments Note You must add a ViiA 7 Instrument to your list before you can manage it 3 After the instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager click Monitor then click Information 5 In the Monitor Instrument screen click Monitor Running Experiment The ViiA 7 Software displays the status attributes calibration status and plot data for the selected ViiA 7 System If a communications warning appears contact your network administrator to troubleshoot the problem You can lose the software connection to the ViiA 7 Instrument if you e Change the ViiA 7 Instrument that is connected directly to your computer e Use the touchscreen to c
263. r during a run e The lamp current is below the acceptable level at the start of the run You cannot proceed with the run until you replace the halogen bulb as explained in Replace the lamp on page 128 e The ViiA 7 Software stopped the run because the lamp current decreased below the acceptable level during the run You cannot proceed with the run until you replace the halogen bulb as explained in Replace the lamp on page 128 Click OK in the message box then replace the lamp bulb e The lamp usage exceeds 2000 hours at the start of a run Click Cancel Run then replace the lamp or click Continue Run Applied Biosystems ViiA 7 Real Time PCR System User Guide 127 Calibration Maintenance Networking and Security 5 Chapter 5 Service Replace the halogen lamp Check the lamp status 1 In the Home screen of the ViiA 7 Software click Instrument Console 2 In the Instrument Console select your ViiA 7 Instrument from the list of instruments then review the Lamp Life and Last Serviced readings in the Maintenance Info pane If the lamp usage is greater than 2000 hours we recommend that you replace the lamp see Replace the lamp below Note The Lamp Life report displays the total number of hours that the halogen lamp has been illuminated The Last Serviced report displays the date that the lamp was installed Replace the lamp IMPORTANT Wear powder free gloves when you handle the lamp
264. r free gloves Safety goggles Note Only the background plate is required for this calibration 384 Well Plate Sample Block 384 Well Region of Interest ROI and Background Plates Centrifuge with plate adapter Powder free gloves Safety goggles Note Only the background plate is required for this calibration Array Card Sample Block Applied Biosystems Array Card filled with Background Mix or deionized water Applied Biosystems Array Card Staker Sealer Centrifuge with centrifuge array card carrier clips and buckets Powder free gloves Safety goggles Pipettor 200 uL with pipette tips When to perform the calibration Perform the background calibration e Monthly or as often as necessary depending on instrument use e After replacing the lamp 48 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the background calibration About the background calibration data During the background calibration the ViiA 7 Software captures a series of images of the background plate using each instrument filter The software compares the fluorescence from each well to the average for the plate A background calibration passes if the collected images for all filters are free of abnormal fluorescence About the data After the calibration you can review the calibration data in the Background tab of the Instr
265. ranscriptase 248 RNase P instrument verification experiment 74 kits 75 outlier removal 79 perform 74 preparation 77 R2 value 79 troubleshoot 83 when to perform 74 robot 27 components 27 28 racks 28 ROI calibration 42 data 43 perform 45 preparation 43 successful 43 troubleshoot 47 when to perform 42 ROX dye 19 62 248 RS232 port 25 28 259 Index run experiments 144 method 249 type 18 S safety Array Card Staker Sealer 38 bar code scanner 229 before operating the instrument 227 biological hazards 234 chemical 231 chemical waste 232 electrical 228 ergonomic 229 guidelines 231 232 233 instrument operation 227 lamp replacement 127 laser 229 moving and lifting 164 227 moving parts 228 physical hazard 228 repetitive motion 229 standards 230 ultraviolet light 228 workstation 229 safety labels oninstruments 15 226 safety standards 230 safety symbols on instruments 224 sample 249 sample block 24 decontamination 124 handling 124 135 installation 135 sample definition file 249 sample file 185 file format 197 scientist user role 107 seal array cards 38 security administrator 101 enable disable 103 instrument setup 156 policies 104 software setup 103 security and auditing 249 260 security administrator account setup 104 disable effect on audit and e sig 103 enable disable 103 export settings 120 export user account settings 120 import settings 120 import user account settings 1
266. rd fails contact Applied Biosystems for further assistance RNase P plate verification run failed Insufficient centrifugation Defective plate seal CAUTION PHYSICAL INJURY HAZARD During AN instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature 1 Unload the RNase P plate or array card from the ViiA 7 Instrument 2 Hold the plate or array card up toa light source and verify that all wells contain the same volume of fluid If there are differences in fluid volumes check the heat seal of the wells with lower volumes for signs of damage or evaporation Also compare the position of the wells that have lower volumes with the outliers that you have removed from the plate If the well positions coincide the heat seal on the plate may be defective resulting in the evaporation of the associated samples 3 Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Instrument does not eject the RNase P plate Adhesive cover may have adhered the plate to the heated cover within the instrument 1 Power off the ViiA 7 Instrument 2 Wait for 15 minutes then power on the ViiA 7 Instrument and eject the plate 3 If the plate does not eject po
267. rds for calibration 38 array cards for verification 74 custom background plate or array card 166 custom dye plate 170 experiments from the instrument 143 custom dye 243 custom dyes 19 62 add to software 171 calibration 168 create plate 170 cycle threshold 242 243 cycling stage 243 D DANGER description 14 data background calibration 49 dye calibration 63 normalization calibration 69 ROI calibration 43 transfer to from the instrument 95 145 uniformity calibration 55 data collection 18 data management 37 date time instrument 152 decontamination identify contaminants 54 sample block 124 delta Rn 243 DHCP support 88 dimensions instrument 20 disable calibration reminders 96 security instrument 156 security software 103 DNS support 88 documentation related 237 door access 24 side 24 dye calibration 60 243 data 63 perform 65 spectra evaluation 64 troubleshoot 68 when to perform 61 Dye Library 243 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security dyes custom 19 62 system 19 62 E electrical protective devices 29 electrical requirements 22 electrical safety 228 electromagnetic compatibility standards See EMC standards electronic signature administrators actions that allow e sig 117 enable or disable 116 functions that require e sig 117 is signed field 122 when security is disabled 116 electronic signature users is signed field 122 sig
268. re displays each operation as it is performed Results export The command used to export the results from experiment files uses the following syntax cmdlineutil exe export parameters The following table lists the acceptable parameters that can be included in any order See Examples on page 190 for and examples of the experiment export command IMPORTANT Enclose file paths in double quotes to allow spaces in the string Parameter Description e lt dirpath gt Required Specifies the path to the directory lt dirpath gt that contains the experiment files eds for which the software exports data Example e D Applied Biosystems ViiA7 Software vl 1 experiments f lt option gt Required Specifies the format of the exported data see page 199 for the export file specifications e ViiA7 Exports data in a format compatible with the ViiA 7 System e SDS23 Exports data in a format compatible with the Applied Biosystems 7900HT Real Time PCR System e RDML Exports data in the real time data markup language RDML format Example RDML l lt path gt Optional Specifies the path lt path gt of the directory to which the software saves the exported files Example 1 bD exports s lt option gt Optional Specifies the data spanning option lt option gt that determines how the software exports data from multiple experiments e sing
269. receding section Hazard symbol English Francais ZN CAUTION Hazardous chemicals Read the Safety Data Sheets SDSs before handling ATTENTION Produits chimiques dangereux Lire les fiches techniques de s ret de mat riels avant toute manipulation de produits CAUTION Hazardous waste Refer to SDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches techniques de s ret de mat riels et la r gulation locale associ es a la manipulation et l limination des d chets AN WARNING Hot lamp AVERTISSEMENT Lampe br lante WARNING Hot Do not remove lamp until 15 min after disconnecting supply AVERTISSEMENT Lampe br lante apr s avoir d connect le c ble d alimentation de l appareil attendre environ 15 minutes avant d effectuer un remplacement de la lampe WARNING Hot Replace lamp with an Applied Biosystems lamp AVERTISSEMENT Composants br lants Remplacer la lampe par une lampe Applied Biosystems CAUTION Hot surface ATTENTION Surface br lante DANGER High voltage DANGER Haute tension WARNING To reduce the chance of electrical shock do not remove covers that require tool access No user serviceable parts are inside Refer servicing to Applied Biosystems qualified service personnel AVERTISSEMENT Pour viter les risques d lectrocution ne pas retirer les capots dont ouverture n
270. rectory lt dirpath gt to which the software saves the new files Example 1 D Applied Biosystems ViiA7 Software vl 1 experiments Before creating experiment files the software confirms whether the export location exists and aborts if the location does not exist m lt filepath gt Optional Specifies the path and name lt filepath gt of the sample file that the software uses to create new files Example m D samplefiles samplefile txt n lt integer gt Optional If the b parameter is not included specifies number of experiments lt integer gt that the software will create If no value is supplied the software creates 25 experiments by default Example n 31 s lt filepath gt Optional Specifies the path and name lt filepath gt of the setup file that the software uses to create new files Example s D setupfiles setupfile txt 188 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference E ViiA 7 Software command line application Parameter Description t lt filepath gt Required Specifies the path and name lt filepath gt of the ViiA 7 Software template file that the software uses to create new files Example t D Applied Biosystems ViiA7 Software vl 1 experiments templatefile edt Optional Configures the software to operate in verbose mode where the softwa
271. red signatures The data must be signed before the user can perform the action Action The ViiA 7 Software requires e signatures when a user Print Report Prints a report from an experiment or study Start Run Initiates a run from the ViiA 7 Software or ViiA 7 Instrument 2 For each selected action enter the number of e signatures from each user role that are required for each meaning before the software can execute the action For example in the following figure at least two users from the Administrator user role must sign an experiment using the Reviewed and Approved Plate Set Up meaning before a user can start the associated run Actions requiring signatures Signature Required Action F Print Report Meaning Administrator Technician Scientist StartRun Reviewed and Approved Plate Set Up 2 o 0 Reviewed and Approved Results ol ol 0 Reviewed and Approved Template ol o 0 3 Click Apply Settings Applied Biosystems ViiA 7 Real Time PCR System User Guide 117 Calibration Maintenance Networking and Security 4 Chapter 4 Security Audit and Electronic Signature Manage electronic signature How the software prompts electronic signature Example 118 If the system is configured to check that data is signed before starting a run or printing a report and the data is not signed the ViiA 7 Software displays a message when the user clicks St
272. rget sample combination Float Automatic Ct Threshold Whether the threshold was determined automatically true or manually false true Or false Ct Threshold The threshold cycle C for the sample at the well Float Automatic Ct Whether the baseline was determined true Or false Baseline automatically true or manually false Baseline Start The first cycle used to calculate the baseline Integer Baseline End The last cycle used to calculate the baseline Integer Custom 1 Custom6 The contents of the custom text fields found in the Results table of the experiment 1024 character string per field If analysis flags are present results data is present in additional columns named for the associated flags true Or false Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 207 208 Appendix E ViiA 7 Software Reference Export formats and file specifications Biological replicate results The following table describes the results data exported from high resolution melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Biogroup The name of the biological replicate group 100
273. s IMPORTANT Balance the loads in opposite buckets in the centrifuge Close the centrifuge cover then spin the array card s for 1 minute at 1200 rpm When the run is finished stop the centrifuge then spin the array card s again for 1 minute at 1200 rpm IMPORTANT Do not try to save time by doing one spin for 2 minutes The two sets of ramps are important for a good fill into the array card When the second run is finished open the centrifuge and check that the fluid levels in the reservoirs of each array card have decreased by the same amount Also check for the formation of bubbles in all wells and note possible problems Correct fill Incorrect partial fill eed oO If necessary centrifuge the array cards for an additional minute to fill any unfilled wells Do not exceed three 1 minute runs or centrifuge the array card for longer than 1 minute at a time Seal the array card s a With the carriage roller assembly of the Array Card Staker Sealer in the Start position place a filled array card into the fixture with the foil side up so that the fill reservoirs are the farthest away from the carriage Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Fill the array cards b Press down on all four corners of the array card to Alignment pins ensure that it is full
274. s product only as specified in this document Using this instrument in a manner not specified by Applied Biosystems may result in personal injury or damage to the instrument CAUTION PHYSICAL INJURY HAZARD The instrument is to be moved and positioned only by the personnel or vendor specified in the applicable site preparation guide If you decide to lift or move the instrument after it has been installed do not attempt to lift or move the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can cause painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons WARNING Do not attempt to lift or move the computer or the monitor without the assistance of others Depending on the weight of the computer and or the monitor moving them may require two or more people Things to consider before lifting the computer and or the monitor e Make sure that you have a secure comfortable grip on the computer or the monitor when lifting e Make sure that the path from where the object is to where it is being moved is clear of obstructions e Do not lift an object and twist your torso at the same time e Keep your spine in a good neutral position while lifting with your legs e Participants should coordinate lift and move intentions with each other before lifting and carrying e Instead of lifting the obj
275. s that you want to restore from the list b Touch Restore to upload the instrument settings from the USB drive IMPORTANT Do not remove the USB drive from the ViiA 7 Instrument until you are instructed to do so Note Alternatively touch Restore Default Settings to restore the ViiA 7 Instrument to the factory settings 5 After the ViiA 7 Instrument reboots unplug the USB drive Perform an instrument self test You can use the ViiA 7 Instrument touchscreen to perform a comprehensive self test of the ViiA 7 Instrument subsystems After the self test is complete the ViiA 7 Instrument generates two files that provide a detailed summary of the instrument condition and function In the event of a problem you can save the results files to a USB drive and email them to Applied Biosystems technical support for a diagnosis Note We recommend running the self test as part of regular maintenance to ensure optimal performance of the ViiA 7 Instrument 1 Ifthe instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press OF 2 Inthe Main Menu touch wx Tools then touch Run Self Test 3 In the Self Test screen touch Start Self Test then wait for the test to complete 4 Optional When the ViiA 7 Instrument completes the self test save the results to a USB drive a Plug a USB drive into the USB port on the right side of the ViiA 7 Instrument touchscreen b To
276. s whether the well was omitted from the true Or false analysis true or included false Tech The number of technical replicates in the Integer Replicates associated biological replicate group RQ The relative quantity calculated for the replicate Float wells of the target sample combination RQ Min The minimum relative quantity calculated for the Float replicate wells of the target sample combination The lower limit of the confidence interval RQ Max The maximum relative quantity calculated for the Float replicate wells of the target sample combination The upper limit of the confidence interval Ct Mean The average Cy of the replicate wells for the Float specified target sample combination Delta Ct Mean The average AC of the replicate wells for the Float specified target sample combination Delta Ct SD The standard deviation of the AC for the replicate Float well Depending on the analysis settings this column may by replaced with Delta Ct SE the standard error of the AC Delta Delta Ct The AAC value of the replicate wells for the Float specified target sample combination Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 215 E Appendix E ViiA 7 Software Reference Export formats and file specifications 7900 export format Exportable files 216 The ViiA 7 Software can export setup and results data from experiment files eds
277. screen click Finish to complete the calibration then click Yes when prompted to save the results 46 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Troubleshoot the ROI calibration Chapter 2 Calibration and Maintenance Perform the ROI calibration Problem symptom Possible cause Action ROI calibration failed ROI image is faint The sample block or heated cover may not be seated correctly 2 Wait for 15 minutes then open the access door 3 Firmly push the sample block and the heated cover Power off and unplug the ViiA 7 Instrument toward the back of the ViiA 7 Instrument to confirm that they are seated correctly IMPORTANT Confirm that the arrows on the front handle of the heated cover align as shown below If the arrows do not align push the heated cover further into the ViiA 7 Instrument until the handle locks into place IMPORTANT Confirm that the indicator on the left side of the sample block is positioned behind the red line on the instrument rail If the indicator is forward of the red line push the sample block into the ViiA 7 Instrument until it is seated correctly SS SS 4 f the ROI calibration continues to fail check the status of the halogen lamp within the ViiA 7 System then replace the lamp if necessary see Replace the halogen lamp on page 127 Instrument does not T
278. se or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided ZN may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTs each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to Applied Biosystems instruments see Safety symbols on page 224 SDSs The SDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day For instructions on obtaining SDSs see SDSs on page 232 IMPORTANT For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer 14 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Safety labels on instruments About This Guide Safety information The following CAUTION WARNING and DANGER statements may be displayed on Applied Biosystems instruments in combination with the safety symbols described in the p
279. ser Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference E Export formats and file specifications Setup file When setup file is selected as an export option the ViiA 7 Software exports sample setup data to a stand alone file The sample setup file describes the sample configuration on the experiment consumable including sample and assay data positions and task assignments File header The file begins with several lines shown in the following table that describe the experiment file and the ViiA 7 Instrument for which it is designed Category Component Output File Version Defines the version of Setup File format used to Integer generate the document Plate Size Defines the number of wells in the plate Integer modeled by the file for example 96 384 Plate ID Defines the ID of the Assay Plate Normally this 100 character string is a bar code printed on the plate Setup File Version lt version number gt xxx Output Plate Size lt number of wells gt Output Plate ID lt plate id gt Assay detector data The assay data describes the qualities of the target assays present on the consumable In the context of the 7900HT System target assays are referred to as detectors The section consists of multiple lines that define the total target assays followed by a column header and tab separated data The first line defines the total num
280. settings After the ViiA 7 Instrument is secured you must enter an administrator password to modify the instrument settings use the firmware tools or deactivate the secure mode IMPORTANT If you enable or disable the ViiA 7 Instrument security auditing and electronic signature feature you must similarly enable or disable the ViiA 7 Software security see page 103 The ViiA 7 Software cannot connect to ViiA 7 Instruments that do not match security settings Note Secure mode limits the number of feature that are available from the ViiA 7 Instrument touchscreen it does not provide user authentication functionality through the instrument touchscreen 1 If the instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press OF 2 In the Main Menu touch Settings then touch Set Administrator Options 3 In the Set Administrator Options screen touch Secure Environment to enable checked or disable unchecked system security 4 Optional To change the administrator password Touch Change Password Enter the current password then touch Done Enter the new password then touch Done Reenter the password when prompted oon FS w Touch OK when prompted Note The default password for the ViiA 7 Instrument touchscreen is password however the password can be changed during installation 5 Touch Save 6 Touch the Administrator Password field enter the administrator pa
281. ssword then touch Done 7 Touch to return to the Main Menu Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix A Manual Instrument Operation A Administrate the instrument from the touchscreen View the instrument log You can use the ViiA 7 Instrument touchscreen to view a log that summarizes instrument activity from the last 6 months For each recorded activity the activity log provides a description of the activity and the date time when it occurred View the log 1 If the instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press OF 2 In the Main Menu touch x Tools then touch View Log 3 In the View Log screen configure the settings to display the records of interest e Select an option from the drop down menu to filter the log e Select Earliest First or Latest First to determine the order to sort the records 4 Touch to return to the Main Menu Applied Biosystems ViiA 7 Real Time PCR System User Guide 157 Calibration Maintenance Networking and Security A Appendix A Manual Instrument Operation Administrate the instrument from the touchscreen 158 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security APPENDIX B Power On or Off Store and Move the ViiA 7 System This appendix covers m Place the ViiA 7 System on standby
282. st excite between 455 and 672 nm and read between 505 and 723 nm Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the dye calibration About the dye The product of a dye calibration is a collection of spectral profiles that represent the calibration data fluorescence signature of each dye standard Each profile consists of a set of spectra that correspond to the fluorescence collected from the wells of the spectral calibration plate The ViiA 7 Software plots the resulting data for each spectral profile in a graph of fluorescence versus filter When the ViiA 7 Software extracts the dye calibration data it evaluates the fluorescence signal generated by each well in terms of the collective spectra for the entire calibration plate Dye spectra are generally acceptable if they peak within the same filter as their group but diverge slightly at other wavelengths see below The ViiA 7 Software can compensate for some differences in a spectral profile by replacing the spectra of unacceptable wells with the spectra of other wells on the reaction plate auto repairing The ViiA 7 Software allows only a few replacements and it may reject the calibration if the spectra between neighboring wells vary significantly Note Because the wells of a calibration plate contain identical concentrations of a dye the resulting signals
283. stallation specification 76 overvoltage category rating 228 P passive reference 246 password administrator 101 changing 121 expiration 104 restrictions 104 pdf action log 111 114 119 audit reports 113 258 perform background calibration 51 dye calibration 65 normalization calibration 71 optical calibration 57 RNase P instrument verification 79 ROI calibration 45 uniformity calibration 57 permissions user account 107 121 physical hazard safety 228 plate background calibration 50 dye calibration 64 normalization calibration 70 RNase P instrument verification 77 ROI calibration 44 56 signing 122 plate adapter 24 installation 139 plate layout 246 plate preparation guidelines 35 plate setup file 246 file format 192 plates calibration 35 pollution requirement 22 port Ethernet 1 25 88 91 RS232 serial 25 28 USB 25 positions robot racks 28 positive control 246 power LED 27 port 25 28 requirements 22 switch instrument 25 power line regulator 29 power on off the instrument 160 161 prepare array cards 38 78 background calibration plate 50 custom dye plate 170 dye calibration plates 64 normalization calibration plate 70 plate for instrument verification 77 RNase P experiment 77 ROI calibration plate 44 56 primer mix 246 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security primer probe mix 246 print action logs 111 114 119 audit re
284. ste safety 233 consumable preparation 35 networking 90 remote monitoring 94 255 Index H halogen lamp See lamp hand held barcode reader 26 hard drive maintenance 37 hazard icons See safety symbols on instruments hazard symbols See safety symbols on instruments hazards See safety heated cover 24 handling 137 installation 137 temperature setting 152 Help system accessing 239 holding stage 244 housekeeping gene 244 humidity requirement 22 icon instrument 152 identifying contamination 54 import 244 audit settings 120 file formats 191 security settings 120 user account settings 120 IMPORTANT description 14 installation category 228 firmware updates 132 halogen lamp 127 heated cover 137 instrument fuses 130 license keys 133 network 88 operating system updates 131 plate adapter 135 139 software 30 software updates 132 specification 76 third party software 31 256 instrument 18 24 25 accessories 176 APIPA support 88 Autodiscovery 154 background calibration 48 control monitor over anetwork 88 data transfer 95 date time setting 152 DHCP support 88 dye calibration 60 168 electrical requirements 22 environmental requirements 22 Ethernet 1 port 88 exhaust venting 22 filter sets 19 fuse replacement 130 heated cover temperature 152 icon 152 installation 164 installation specification 76 IPV4LL 88 layout and connections 23 log 157 maintenance 34 147 maintenance reminders 153 mDNS DNS su
285. sults data depends on the experiment type and the analysis settings The section begins with a column header followed by the results data where each row contains the data for a single well separated by tab characters If a well contains more than one assay target the Vii A 7 Software lists the data for each additional assay on separate rows repeating the well number and sample information This section describes the following results data formats Standard curve relative standard curve and comparative CT 207 GeNOLY PING erdie Nea ahi eR aN wera EE oP aia wte aa hed wears 210 Me J Hg CUR VG se aiecs entre deca gated aceite demain od adhe E EEE E EEAS EE 211 Presence absence sinri reira eee eee eee e eee e eee e ee eees 212 LUC ye Catals 5 kiss Maen ses ais Gate ited Tete a Gane a e ea 213 Technical replicate results 0 0 006 c cen es 209 Technical analysis result Study n 6 ccc eens 214 Biological replicate results 6 66 eens 208 BioGroup analysis results study 0 cece eee ee 215 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference Export formats and file specifications The following table describes the results data exported from standard curve relative standard curve and comparative Cy experiments Because columns can be omitted from the results the exported file may contain a s
286. system 22 cece eee nrnna 103 Access the Security screen and enable or disable security 103 Set account setup and security policies 0 0 0c e cece ee eee eee eee 104 Set up messaging notifications 00 eee eee eee eee 105 Manage lser accounts a 2 26 bee le bend pi dea ee eee etn 106 Create or edit a user account cece cece ene een e enna 106 Determine the name of the logged in user 2200000 cece eee eee eee 107 Create or edita user role is 5 ce eae deeded Mae ened 107 View and print a user report 2 0c c ect rrr e eae 109 ManageaUditingns cit tte Litera eet nea ice oS a eel ey leet tele aaea AEE 110 Access the Audit screen and enable or disable auditing 110 Select objects to audit 2 2 2 e eee e enna 110 Create audit reason settingS 0 0 rnrn rnnr rnrn eeeees 110 Generate audit reports 000 c cece cee eee ene e teen eeeees 111 Display audit histories from the Security Settings dialog box 111 Display audit histories for an experiment template or study 114 Manage electronic Signature 0 00 c cece eee eee eee eee eeees 116 Access the e Signature Settings screen and enable or disable e sig 116 Configure the meanings of the electronic signatures 0000eeeee 116 Configure the e signature rights for user roles 00000e
287. t www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition http www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix F Safety F Safety alerts Safety alerts For the definitions of the alert words IMPORTANT CAUTION WARNING and DANGER see Safety alert words on page 14 General alerts for all chemicals Avoid contact with skin eyes and or clothing Read the SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves General alerts for instrumentation CAUTION Before using a cleaning or decontamination method other than those recommended by the Applied Biosystems verify with Applied Biosystems that the proposed method will not damage the equipment N WARNING This instrument is designed for 12 V 75 W halogen lamps only Specific alerts for instrumentation CAUTION FIRE HAZARD For continued protection against the risk of fire replace fuses only with listed and certified fuses of the same type and rating as those currently in the instrument CAUTION PHYSICAL INJURY HAZARD Do not attempt to lift the instrument or any other heavy objects unless
288. t nucleic acid sequences targets using real time analysis e Qualitative research detection of targets using post PCR endpoint analysis e Qualitative analysis of the PCR product achieved by melt curve analysis that occurs post PCR About data collection The Applied Biosystems ViiA 7 Real Time PCR System collects raw fluorescence data at different points during a PCR depending on the type of run that the ViiA 7 Instrument performs Run type Data collection point Real time runs Standard curve Relative standard curve Comparative Cy AAC7 Melting curve The ViiA 7 Instrument collects data following each extension step of the PCR Post PCR endpoint runs Genotyping Presence absence The ViiA 7 Instrument collects data 1 Before the PCR For presence absence experiments data collection before the PCR is optional but recommended 2 Optional During the PCR The ViiA 7 Instrument can collect data during the run real time collecting data during the run can be helpful for troubleshooting 3 After the PCR Regardless of the run type a data collection point or read on the Applied Biosystems ViiA 7 Real Time PCR System consists of three phases 1 Excitation The ViiA 7 Instrument illuminates all wells of the reaction plate within the instrument exciting the fluorophores in each reaction 18 Applied Biosystems ViiA 7
289. t permitted to enter a reason 2 As needed click Create or select a reason from the list then click Edit or Delete 110 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators 4 Generate audit reports Generate audit reports You can use the ViiA 7 Software to generate reports of audit history from both the Security Settings dialog box and open experiments templates or studies m Display audit histories from the Security Settings dialog box 111 m Display audit histories for an experiment template or study 114 Display audit histories from the Security Settings dialog box Display audit 1 In the ViiA 7 Software select Tools gt Security gt Settings histories 2 In the Security Settings dialog box select the Audit tab then click View Reports Note To access the Audit Reports screen the user role for an account must specify the Configure SAE permission Users without the Configure SAE permission can view object audit histories for individual entries in the libraries by selecting entries then clicking View Audit History 3 Select a tab to display e System Configuration History Security audit and electronic signature configuration records including audit history for each user account e Action Record System specified audit events 4 Optional Select Filter by then filter the table
290. t provide adequate detail for all network architectures Note The following procedure is valid for the Microsoft Windows XP operating system 1 Use the Ethernet cable to connect the computer to the nearest network port 2 Power on the computer then log in using a user account that belongs to the Administrators user group 3 In the computer desktop right click My Network Places then select Properties 4 Right click Local Area Connection then select Properties 5 Select Internet Protocol TCP IP then click Properties 92 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 3 Networking 3 Connect the computer to the network 6 Set the Internet Protocol TCP IP Properties for either DHCP or Static IP communication eewanr Action configuration DHCP 1 Select Obtain an IP address automatically 2 Set the DNS address If the computer obtains DNS addresses e Automatically Select Obtain DNS server address automatically e Statically Select Use the following DNS address enter the address of the preferred and alternate DNS servers if available Static IP 1 Select Use the following IP address 2 In the IP Address field enter the static IP address 3 If necessary enter a subnet mask 4 If necessary enter a static gateway address in the Default Gateway field 7 If your network requires advanced TCP IP setup such
291. t the calibration Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the uniformity calibration Perform the uniformity calibration The uniformity calibration generates data that allows the ViiA 7 Software to compensate for the physical effects of the ViiA 7 System filters Materials required See Perform the ROI calibration on page 42 for a complete list of materials for the calibration When to perform the calibration Perform a uniformity calibration every 6 months or as often as necessary depending on instrument use About the uniformity calibration data During the uniformity calibration the ViiA 7 Software captures a series of images of the ROI plate using each instrument filter After the calibration you can review the data in the Uniformity tab of the Instrument Manager The Analysis Data plot left side displays the fluorescence data in all filters The Well Table tab right side displays the data collected for the current calibration in all well positions The QC tab displays a summary of quality check performed by the ViiA 7 Software on the calibration data Applied Biosystems ViiA 7 Real Time PCR System User Guide 55 Calibration Maintenance Networking and Security 2 Chapter 2 Calibration and Maintenance Perform the uniformity calibration Prepare the calibration plate or array
292. tatus reports WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can be heated to 100 C Before removing the plate wait until it reaches room temperature IMPORTANT If the ViiA 7 Instrument does not eject the plate remove the plate as explained in Troubleshoot the background calibration on page 53 8 Discard or store the plate or array card Consumable Action Array card Discard the array card Plate Return the background plate to its packaging sleeve then return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate is stored in its original packaging sleeve you can use it to calibrate a ViiA 7 System 3 times for up to 6 months after you open it 9 Inthe Background Calibration screen click Finish to complete the calibration then click Yes when prompted to save the results 52 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the background calibration Troubleshoot the background calibration Problem symptom Possible cause Action Background calibration failed One or more wells of the background plate produced spectra that exceed the maximum limit for the ViiA 7 Instrument Repeat the calibration using the same background plate If the calibration fails again
293. te To perform an instrument verification 1l2lsl a s e z s ol1o 11 1213 141516171819 20 21 22 23 24 PORT run each solution is loaded into the empty array card in the arrangement shown right When complete the array card contains five replicate groups of standards 200 400 800 1600 and 3200 copies two of unknown populations 800 and 1600 copies and one that serves as a no template control NTC olz a 0 m o o a gt Applied Biosystems ViiA 7 Real Time PCR System User Guide 75 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Verify the instrument performance About the analysis The ViiA 7 Software performs the same analysis of data from an instrument verification run regardless of the type of consumable used 96 384 well plate or array card After the run the ViiA 7 Software 1 Generates a standard curve from the averaged threshold cycle Cr values of the replicate groups of standards Calculates the concentration of the two unknown populations using the standard curve Calculates the following to assess the ViiA 7 System performance CopyUnk 3 0CopyUnk2 gt CopyUnk 3 6copyunk1 where e CopyUnk Average copy number of unknown population A OCopyUnk1 Standard deviation of unknown population A e CopyUnk Aver
294. tenance Networking and Security Glossary region of interest ROI calibration regression coefficients regression line reject well relative standard curve method replicate group replicates reporter reverse primer reverse transcriptase Rh ROX dye rs number 248 Type of calibration in which the software maps the positions of the wells on the sample block of the instrument The software uses the ROI calibration data to associate increases in fluorescence during a run with specific wells of the plate A calibration image for each individual filter must be generated to account for minor differences in the optical path Values calculated from the regression line in standard curves including the R value slope and y intercept You can use the regression coefficients to evaluate the quality of results from the standards See also standard curve In standard curve and relative standard curve experiments the best fit line from the standard curve Regression line formula Cr m log Qty b where m is the slope b is the y intercept and Qty is the standard quantity See also regression coefficients An action that the software performs during analysis to remove one or more wells from further analysis if a specific flag is applied to the well An experimental method to determine relative quantities This method compensates for target and endogenous control efficiency differences within each run In
295. the sample block 10 Firmly press down on the handle of the sample block then remove it from the ViiA 7 Instrument Place the sample block on a clean dry surface Install the new sample block into the ViiA 7 Instrument IMPORTANT After installing the sample block confirm that the indicator on the left side of the sample block is positioned behind the red line on the instrument rail If the indicator is forward of the red line push the sample block into the ViiA 7 Instrument until it is seated correctly If you are installing a sample block of a different consumable format replace the heated cover and plate adapter if necessary to match the new consumable format IMPORTANT If you are installing a sample block of a different format you must also change the plate adapter to match the new consumable format Close the access door IMPORTANT Confirm that the access door is completely closed The ViiA 7 Software displays an error message if the door is not completely closed and latched or if the sample block is not seated correctly Plug in and power on the ViiA 7 System In the Home screen of the ViiA 7 Software click Instrument Console In the Instrument Console select your ViiA 7 Instrument from the list of instruments then review the Block Type field in the Instrument Properties pane The installation is successful if the ViiA 7 Instrument powers on and if the Block Type field displa
296. ther the allele 1 threshold was determined true Or false Automatic Ct automatically true or manually false Threshold Allele1 The start cycle used to calculate the baseline Float Baseline Start section of allele 1 Allele The end cycle used to calculate the baseline Float Baseline End section of allele 1 Allele2 Whether the allele 2 threshold was determined true Or false Automatic Ct automatically true or manually false Threshold Allele2 The first cycle used to calculate the baseline for Float Baseline Start allele 2 Allele2 The last cycle used to calculate the baseline for Float Baseline End allele 2 Custom 1 The contents of the custom text fields found inthe 1024 characterstring Custom Results table of the experiment per field Well numbers start at 1 for well A1 upper left corner and increase from left to right and from top to bottom Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix E ViiA 7 Software Reference Export formats and file specifications Melting curve The following table describes the results data exported from melting curve experiments Because columns can be omitted from the results the exported file may contain a subset of the data columns below Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Sample Name The name of the sample contained b
297. thernet 1 port to the network interface card in the computer IMPORTANT Do not use a standard Ethernet cable to connect the ViiA 7 Instrument to the computer IMPORTANT Do not connect the Ethernet cable to the Ethernet 2 port on the ViiA 7 Instrument The second port is for Applied Biosystems service use only Install the sample block and heated cover assemblies Perform a RNase P instrument verification run If the run Passes Do not recalibrate the ViiA 7 System No further action is necessary Fails Perform the following calibrations in the specified order ROI background uniformity dye then normalization calibrations Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security APPENDIX C Creating Custom Calibration Plates and Array Cards This appendix covers m Create a background plate or array card 6 eee 166 m Create a custom dye plate for calibration 00 eee eee ee 168 Applied Biosystems ViiA 7 Real Time PCR System User Guide 165 Calibration Maintenance Networking and Security 6 Appendix C Creating Custom Calibration Plates and Array Cards Create a background plate or array card Create a background plate or array card Materials required 96 384 Well Plate Sample Block Array Card Sample Block Whenever possible use a Background Plate or the TaqMan Array Background Buffer that
298. thers from exposure to the beam The optional hand held bar code reader functions as an extension of the keyboard that you can use to scan bar codes into the ViiA 7 Software To scan a bar code using the hand held bar code reader 1 Select the field in the ViiA 7 Software where you want to enter the bar code 2 Hold the hand held bar code reader 20 to 30 cm away from a plate and aim at the center of the bar code then press the trigger Slowly move the scanning beam across the bar code until the reader emits a high pitched tone When the reader scans a bar code it automatically e Transmits the alphanumeric equivalent of the bar code to the ViiA 7 Software The software enters the bar code text wherever the cursor is active e Transmits a carriage return character the equivalent of pressing the Enter key For more information on the hand held bar code reader see the bar code reader user documentation shipped with the ViiA 7 System 26 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 1 Getting Started 1 ViiA 7 System hardware Twister IlI Robot components Front view Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security The ViiA 7 System supports the use of the Applied Biosystems Twister II Robot an optional ViiA 7 System accessory that consists of the components shown b
299. tion Dye calibration plate was centrifuged insufficiently Dye calibration plate contains old or insufficient reagents If you are running a custom dye calibration plate the dye may not be present at a sufficient concentration 1 Unload the ViiA 7 System and view the wells of the dye calibration plate If the liquid in the wells is not e At the bottom of the wells centrifuge the plate for a longer time then repeat the calibration e Equivalent in volume the plate is not sealed and J the reagents have evaporated Discard the plate and run another 2 If the dye calibration plate appears to be normal discard the plate and run another 3 If the problem persists contact Applied Biosystems If you are running a custom dye calibration plate create another plate but increase the concentration of the dye that produced insufficient signal Spectra contain peaks in more than one filters One or more raw spectra exceed the maximum limit for the ViiA 7 System Fluorescent contaminants are present on the sample block or dye calibration plate If you are running a custom spectral calibration plate the dye may be too concentrated Verify that contaminants are not present by performing a background calibration see Perform the background calibration on page 48 If the background calibration does not show sample block contamination the dye calibration plate may be contaminated
300. tion and or inconsistent reaction volumes Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit Repeat the experiment and make sure to properly seal and centrifuge the RNase P plate or array card If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Well displays the BLFAIL flag indicating the software cannot calculate the best fit baseline for the data Amplification too late No amplification Well displays the CTFAIL flag indicating the software cannot calculate the threshold cycle C Well displays the EXPFAIL flag indicating the software cannot identify the exponential region of the amplification plot Amplification too early Amplification too late Low amplification No amplification If a well is flagged confirm the results 1 Select the flagged well s in the plate layout or well table 2 View the amplification plot R vs Cycle and DR vs Cycle and check for early late low or no amplification Contact Applied Biosystems to order a replacement TaqMan RNase P plate or array card kit Repeat the experiment making sure to properly seal and centrifuge the RNase P plate If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Well displays the HIGHSD flag indicating the C standard deviation for the replicate group exceeds the current f
301. tions in spectral position and peak position are caused by minor differences in the optical properties and excitation energy between the individual wells Select the QC tab then review the summary of wells that failed the quality check QC Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the dye calibration 9 After you inspect the dye spectra verify the status of the calibration e Analysis Status Indicates the success of the calibration where passed indicates that the run produced viable calibration data and failed indicates that the run did not produce data or that the data it did collect is unusable e QC Status Indicates the quality of the calibration data where passed indicates that all wells produced data that passed the quality check and failed indicates that one or more wells produced dye spectra that differ significantly from the other wells on the plate Analysis ac status status Action Passed Passed 1 Click Next 2 Enter any comments you have in the Comments field click Finish then click Yes when prompted to save the results 3 Remove the plate or array card when the ViiA 7 Instrument ejects the tray arm Passed Failed Troubleshoot the failed calibration as described in Troubleshoot the dye calibration on page 68 Note You can accept a calibration that pass
302. to the fill port There is a fill port on the left arm of each fill reservoir the larger of the two holes Fill Vent A port port 1 2 3 4 5 6 O O O AAN E d Dispense the fluid so that it sweeps in and around the fill reservoir toward the vent port When pipetting the reagents into the array card pipette the entire 100 uL volume into the fill reservoir but do not go past the first stop of pipettor plunger or you may blow the solution out of the port IMPORTANT Do not allow the tip to contact and possibly damage the coated foil beneath the fill port 5 Repeat step 4 to fill the remaining array card with the appropriate calibration reagents Applied Biosystems ViiA 7 Real Time PCR System User Guide 39 Calibration Maintenance Networking and Security Z 40 Chapter 2 Calibration and Maintenance Fill the array cards 10 11 Place the filled array card s into a centrifuge array card carrier clip and place empty array cards in the remaining slots Confirm that the labels on the buckets and clips face the same way Place the filled carrier clips into the centrifuge buckets Make sure that the array card fill reservoirs and bucket and clip labels face outward when loaded into the centrifuge IMPORTANT You must run the centrifuge with all four buckets in place and each of the two carriers filled with array cards Place empty array card into unfilled slot
303. to provide a user name and password when performing certain functions Depending on the way that your administrator configures these features you may see the following dialog boxes and prompts when you use the software If security is enabled on your system you must provide a user name and password to access the software Your access to functions in the software is based on the permissions associated with your user account Functions for which you do not have permissions are grayed Note If the ViiA 7 Software is configured for password expiration you will periodically be prompted to change your password Note If the ViiA 7 Software is configured to monitor failed log in attempts you will be locked out of the software if you incorrectly enter your user name or password for a specified number of times If your user account does not have permission to perform any function in the software menu commands are grayed Change your password when it expires When your password is about to expire a message is displayed when you log in To change your password select Tools Change Password Enter your current password then enter the new password two times then click OK Applied Biosystems ViiA 7 Real Time PCR System User Guide 121 Calibration Maintenance Networking and Security Chapter 4 Security Audit and Electronic Signature Audit Account suspension Session time out Audit If the ViiA 7 Software is con
304. to view details for individual records in the table on the bottom right In the ViiA 7 Software open an experiment eds or template edt file In the open experiment template or study click o Audit In the table on the left select the records to be exported e Click in the table then press Ctrl A to select all the records in the table e Click and drag or press Shift to select continuous rows e Press Ctrl to select discontinuous rows Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Section 4 1 Administrators 4 Generate audit reports e Click Export Summary to export only the records in the left hand table e Click Export Details to export the records in the left hand table and the associated details 4 Export the records 5 Select a location for the export file enter a name for the file then click Save 6 Click OK in the confirmation message Print audit records 1 In the ViiA 7 Software open an experiment eds or template edt file 2 In the open experiment template or study click Audit 3 Click View Report to open the Print Preview dialog box 4 Preview save or print the report e Click H Save to save the report as a pdf or html file Enter the file name select a Tacanan select the file type then click Save e Click h Print to send the report to the printer In the Print dialog box select the printer and print o
305. trument are categorized as Class 2 classification II lasers Laser safety Class 2 II lasers are low power visible light lasers that can damage the eyes Never requirements look directly into the laser beam The scanner is designed to prevent human access to harmful levels of laser light during normal operation user maintenance or during prescribed service operations WARNING LASER HAZARD Class 2 II lasers can cause damage to eyes Avoid looking into a Class 2 II laser beam or pointing a Class 2 II laser beam into another person s eyes Workstation safety Correct ergonomic configuration of your workstation can reduce or prevent effects such as fatigue pain and strain Minimize or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions CAUTION MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by potential risk factors that include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors To minimize musculoskeletal and repetitive motion risks e Use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse e Position the keyboard mouse and monitor to promote relaxed body and head postures Applied Biosystems ViiA 7 Real Time PCR System User Guid
306. ubset of the data columns below Standard curve relative standard curve and comparative Cy Column Description Output Well The number of the well on the consumable Integer 1 to 96 384 Sample Name The name of the sample contained by the well 100 character string Target Name The name of the target assay added to the well 100 character string Task The task assigned to the target in the well UNKNOWN NTC Or STANDARD Reporter The reporter dye that labels the probe for the target assay 100 character string Quencher The quencher dye that labels the probe for the target assay 100 character string CT The calculated threshold cycle Cy for the target at the specified well Float Ct Mean The average Cy of the replicate wells for the specified target sample combination Float Ct SD The standard deviation of the average Cy of the replicate wells for the specified target Float Quantity e Unknown wells The calculated quantity for the sample at the well e Standard wells The quantity assigned to the standard at the well Float Quantity Mean e Unknown wells The average quantity of the replicate wells for the target sample e Standard wells The quantity assigned to the replicate wells for the target sample Float Quantity SD The standard deviation of the average quantity of the replicate wells for the ta
307. uch Save to USB IMPORTANT Do not remove the USB drive from the ViiA 7 Instrument until instructed to do so c When the ViiA 7 Instrument finishes writing the results to the USB drive touch OK then remove the USB drive 5 Touch WW to return to the Main Menu a Applied Biosystems ViiA 7 Real Time PCR System User Guide 149 Calibration Maintenance Networking and Security A Appendix A Manual Instrument Operation Maintain the instrument from the touchscreen Update the instrument firmware You can download ViiA 7 Instrument firmware updates directly from the service section of the Applied Biosystems website After obtaining a firmware update transfer the update to the ViiA 7 Instrument using a USB drive Update the firmware 1 Download the firmware update a Go to https www 2 appliedbiosystems com support software b In the Software Downloads page select Applied Biosystems ViiA 7 Real Time PCR System from the menu c In the Software Downloads page for your ViiA 7 Instrument click Updates Patches d Download the ViiA 7 Instrument firmware to a USB drive 2 Plug the drive into the USB port on the right side of the ViiA 7 Instrument touchscreen 3 If the instrument is in standby touch the ViiA 7 Instrument touchscreen to awaken it then press OF 4 Inthe Main Menu touch Tools then touch Upgrade Firmware 5 In the Upgrade Firmware s
308. uide Calibration Maintenance Networking and Security Appendix F Safety F Instrumentation safety Environmental The following symbol applies to all Applied Biosystems electrical and electronic symbols on products placed on the European market after August 13 2005 instruments Symbol Description Do not dispose of this product as unsorted municipal waste Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of waste electrical and electronic equipment WEEE Call your local Applied Biosystems Customer Service office for equipment pick up and recycling See www appliedbiosystems com for a list of customer service offices in the European Union _o6 European Union customers a Applied Biosystems ViiA 7 Real Time PCR System User Guide 225 Calibration Maintenance Networking and Security F Appendix F Safety Instrumentation safety Locations of safety labels on instruments The ViiA 7 Instrument contains warnings at the locations shown below I My i i mi iR 226 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix F Safety Instrumentation safety General instrument safety Moving and lifting the instrument Moving and lifting stand alone computers and monitors Operating the WARNING PHYSICAL INJURY HAZARD Use thi
309. uipment for the Heating of Materials EN 61010 2 081 2002 A1 2003 Particular Requirements for Automatic and Semi Automatic Laboratory Equipment for Analysis and Other Purposes EMC The ViiA 7 Real Time PCR System meets European requirements for emission and immunity EMC Directive 2004 108 EC EN 61326 1 2006 Electrical equipment for measurement control and laboratory use Part 1 General EMC requirements Group 1 Class B This instrument has been tested to and complies with standard AS NZS 2064 Limits and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radio frequency Equipment Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix F Safety F Chemical safety Chemical safety General chemical safety Chemical hazard WARNING CHEMICAL HAZARD Before handling any chemicals refer to warning the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD All chemicals in the instrument are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can aX crack and leak Each 4 liter
310. ument then close the access door IMPORTANT After installing the sample block confirm that the indicator on the left side of the sample block is positioned behind the red line on the instrument rail If the indicator is forward of the red line push the sample block into the ViiA 7 Instrument until it is seated correctly Close the access door IMPORTANT Confirm that the access door is completely closed The ViiA 7 Software displays an error message if the door is not completely closed and latched or if the sample block is not seated correctly Plug in then power on the ViiA 7 System Perform a background calibration to confirm that you have eliminated the contamination If the contamination remains repeat steps 2 through 5 then clean the contaminated wells of the sample block using a 95 ethanol solution a Pipette a small volume of 95 ethanol solution into each contaminated well b Ineach contaminated well pipette the solution up and down several times to rinse the well c Pipette the ethanol solution to a waste beaker Repeat steps 5 through 9 to rinse the wells of the sample block and to verify that you have eliminated the contamination IMPORTANT Always use deionized water to rinse wells after cleaning with bleach or ethanol solution If the contamination remains repeat steps 2 through 5 then clean the contaminated wells of the sample block using 10 bleach solution a Pipette a small v
311. ument Manager The Analysis Data plot left side displays the fluorescence data in all filters The Well Table tab right side displays the data collected for the current calibration The QC tab displays a summary of quality check performed by the ViiA 7 Software on the calibration data Background Fluorescence data collected by the ViiA 7 Instrument includes a fluorescence signal fluorescence inherent to the system referred to as background fluorescence Background fluorescence is a composite signal found in all spectral data that consists of fluorescence from several sources including e Background electronic signal e Contaminants in the sample block e The plastic consumable plate or array card Prepare the background plate or array card IMPORTANT Wear powder free gloves and safety glasses when you prepare plates or array cards Prepare the background calibration consumable appropriate for your instrument m Prepare the background calibration plate 00 6 c eee 50 E Fillthe array Cards sips 0006s cccsaneesawneesmenes eee tees EEE E 38 Applied Biosystems ViiA 7 Real Time PCR System User Guide 49 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Perform the background calibration Prepare the 1 Remove the background plate from the freezer then allow it to warm to room background temperature approximately 5 minutes calibration plate 2 Remove the background
312. ument from the list of instruments on the network then click Add to My Instruments Note You must add a ViiA 7 Instrument to your list before you can manage it 3 After the ViiA 7 Instrument is added to your list select it then click Manage Instrument 4 In the Instrument Manager start the calibration wizard a Click Maintenance then click Dye b In the Dye Calibration screen select System Dye Calibration then click Start Calibration 5 In the Dye Calibration screen select the dye to calibrate from the Dye Name drop down list then perform the calibration as instructed 6 Load the calibration plate or array card into the ViiA 7 Instrument a Confirm that the dye plate or array card that you are about to load matches the dye selected in the ViiA 7 Software The name of the dye contained by the consumable is next to the bar code on the front of the plate or array card b Load the dye plate or array card into the plate adapter Ensure that the plate or array card is properly aligned in the holder e A Load 96 384 well plates with the A1 position at the top left corner of the plate adapter e B Load both plates and array cards with the bar code facing the front of the instrument IMPORTANT Plates should be loaded and unloaded by operators who have been warned of the moving parts hazard and have been adequately trained Applied Biosystems ViiA 7 Real Time PCR System User Guide 6
313. unusu eee eens mM View the instrument log 6 6 Note The touchscreen does not provide access to all instrument functions Features such as instrument calibration and remote notification are available only through the ViiA 7 Software Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security 151 Appendix A Manual Instrument Operation Administrate the instrument from the touchscreen Define the date and time If the instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press Ot In the Main Menu touch Settings then touch Set Date amp Time In the Set Date amp Time screen a Touch the Time zone field then touch the correct time zone from the list b Touch the Date field enter the current date then touch Done Touch the Date Format dropdown list then select the format for your region Touch each Time field enter the appropriate time units then touch Done Touch 12 Hour or 24 Hour to select the appropriate time format m o Qa A Touch Save to save the settings then touch OK when prompted 4 Touch to return to the Main Menu Define the instrument settings 152 1 If the instrument is in standby touch the ViiA 7 System touchscreen to awaken it then press In the Main Menu touch Settings then touch Configure the Instrument Touch the Instrument Name field enter up to a 16 character name for
314. uorescent contamination on the plate or array card or sample block e QC Status Indicates the quality of the calibration data where passed indicates that all wells produced data that passed the quality check and failed indicates that one or more wells produced spectra that deviate significantly from the other wells on the plate Analysis ac Action status status Passed Passed Click Next then remove the plate or array card when the ViiA 7 Instrument ejects the tray arm Passed Failed Troubleshoot the failed calibration as described in Troubleshoot the uniformity calibration on page 59 Failed Failed Note You can accept a calibration that passes the Analysis Status check but fails the QC Status check We recommend using calibrations that yield passing results for both status reports WARNING PHYSICAL INJURY HAZARD During instrument operation the plate can be heated to 100 C Before removing the plate wait until it reaches room temperature IMPORTANT If the ViiA 7 Instrument does not eject the plate remove the plate as explained in Troubleshoot the uniformity calibration on page 59 8 Discard or store the plate or array card Consumable Action Array card Discard the array card Plate Return the ROI calibration plate to its packaging sleeve then return the packaged plate to the freezer IMPORTANT Do not discard the calibration plate If the plate
315. urce and location of the contamination 1 While viewing the background calibration data in the Analysis screen select the QC tab and review the list of wells that failed the quality check Rotate the background plate 180 then perform the background calibration again Determine the location of the contaminated wells again If the position s of the contaminated well s in step 1 and step 2 are e Identical The sample block is contaminated Decontaminate the sample block see Decontaminate the sample block on page 124 e Reversed The background plate or array card is contaminated Discard the plate or array card then perform the background calibration using a new background plate or array card If the calibration fails after you replace the background plate and decontaminate the sample block a Cover a plate or array card with a piece of black paper b Perform the background run as explained in this chapter substituting the plate or array card covered with paper for the background plate or array card c After the run is complete and while viewing the calibration data select all wells in the Plate Layout tab then view the Spectral plot for the peak s If the peak associated with the contamination is e Visible The optics of your ViiA 7 System may be contaminated Contact Applied Biosystems for further support e Absent The sample block is contaminated Decontaminate the sample block again and repea
316. urity Audit and Electronic Signature Generate audit reports Export audit records You can export audit records to a txt file for additional manipulation and reporting outside the ViiA 7 Software 1 2 Display the records of interest as described above Click Export Specify a name and location for the export txt file then click Save Note If you export audit records for samples that are not in their original location samples have been deleted or moved an error message is displayed Return sample data files to their original location then export again Display audit histories for an experiment template or study Display the audit 1 history 2 3 4 Export audit records 1 114 In the ViiA 7 Software open an experiment eds template edt or study edm file In the open experiment template or study click o Audit then click Audit Records Studies only Select the audit records of interest e Study to view the audit records for the study e The name of an experiment to view the audit records for the experiment Optional Filter the table To view fewer records a Check the Filter by check box b Enter criteria for the records of interest such as a date range a user name or a type of action c Click Refresh To view details for a specific record a Click the row in the list on the left to view the details of the record in the table on the upper right b Click any row
317. ut 00 c eee eee eens 20 ViiA 7 System specifications 2 0 0 cence nee nee nne 20 ViiA 7 System layout and connections 0 0 ccc cece eee neces 23 ViiA 7 System hardware 0 00 c cece cece cece ence e nen n een e nn eenes 24 Instrument components 000 cece eee eee eens 24 Barcode readers sce 42 ayels adra anan inie RAAR hs cada abe iee ad tats 26 Twister Il Robot components 0c c cece cence nee n een eens 27 Electrical protective devices 000 c eect tee eeeeeee 29 VIA 7 System software sensat d dapuran Ena cen nnn nnn ene ne en eens 30 Computer requirements 0000 cece cece eee ete e tee eee etnies 30 Software installation 2 0 0 0 ccc cece nen nett teen eee 30 Third party software 00 cece eee eee eee eee eaes 31 Using this Guide ccsssies s caging 2S acne Se ngs De Soa EARS SA OER Re aes 32 Applied Biosystems ViiA 7 Real Time PCR System User Guide 5 Calibration Maintenance Networking and Security Contents CHAPTER 2 Calibration and Maintenance 002eeeeeeee ee eeeeees 33 Calibrate and maintain the ViiA 7 System 2 0 00 nunna cece eee eee ene 34 Recommended calibration and maintenance schedule 00e eee eee 34 About the consumables osse densi 0 00 cece ene ene een eee eeaas 35 Perform regular data maintenance 000cee cece eee eet eet eeeeees 37 Maintain the computer hard drives
318. ves while creating the calibration array cards Note This procedure explains how to create all of the array cards required to calibrate the ViiA 7 System but not all of them are required for a monthly maintenance Before preparing array cards for calibration see Recommended calibration and maintenance schedule on page 34 to determine which calibrations are required Note You can view a video of the array card loading procedure on the Applied Biosystems website To view the demonstration go to www2 appliedbiosystems com lib multimedia taqman_tlda tlda_1 cfm 1 38 Remove the tubes of calibration solutions from 20 C allow them to thaw then vortex the tubes to mix the contents well Remove the Applied Biosystems Array Cards from their box and place them ona clean dry surface Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance 2 Fill the array cards 3 Using a permanent marker mark the side of the empty array cards with e Background e FAM e ROI e ROX e VIC e FAM ROX e VIC ROX 4 For each array card pipette 100 uL of the appropriate calibration solution into each of the eight reservoirs in the array card a Place the array card on a lab bench with the foil side down b Load 100 uL of the calibration solution into a pipette c Hold the pipette in an angled position 45 degrees and place the tip in
319. wer off and unplug the ViiA 7 Instrument then open the access door 4 Wearing powder free gloves reach into the ViiA 7 Instrument and remove the plate from the heated cover then close the access door 5 Perform a background calibration to confirm that the sample block has not been contaminated Negative control well displays the AMPNC flag indicating that the well amplified Contamination in one or more PCR reaction components contained in the negative control well Contact Applied Biosystems to order a replacement RNase P plate or array card kit If the replacement RNase P plate or array card fails contact Applied Biosystems for further assistance Applied Biosystems ViiA 7 Real Time PCR System User Guide 83 Calibration Maintenance Networking and Security Chapter 2 Calibration and Maintenance Verify the instrument performance Problem symptom Possible cause Action Well displays the BADROX flag indicating the passive reference signal is unacceptable for the normalization of the reporter dye signal Droplets on the sides of the wells Improper sealing or seal leaks Condensation on the reaction plate If a well is flagged confirm the results 1 Select the flagged well s in the plate layout or well table 2 View the amplification plot R vs Cycle and review the data in the C region for abnormalities Examine the reaction plate to check for condensa
320. wing table describes the safety symbols that may be displayed on Applied Biosystems devices Each symbol may appear by itself or with text that explains the relevant hazard These safety symbols may also appear next to DANGERS WARNINGS and CAUTIONS that occur in the text of this and other product support Description Description Indicates that you should proceed with appropriate caution and consult the product insert for further information If a product insert does not exist or if the product insert does not contain the symbol or the required information consult the user manual Indicates the presence of a pinching hazard and to proceed with appropriate caution Indicates the presence of moving parts and to proceed with appropriate caution Indicates the presence of a biological hazard and to proceed with appropriate caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a laser light in the instrument and to proceed with appropriate caution Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution gt bebei Indicates the presence of an ultraviolet light and to proceed with appropriate caution Electrical symbols on instruments Symbol Safety symbols documents Symbol 224 Applied Biosystems ViiA 7 Real Time PCR System User G
321. xport formats File format Description See ViiA 7 export file A ViiA 7 formatted text file that contains setup and or page 200 results data exported from an experiment file eds 7900 export file A legacy 7900 formatted text file that contains setup page 216 and or results data exported from an experiment file eds RDML export file A compressed XML file that contains setup and or page 222 results data exported from an experiment file eds and parsed in Real time PCR Data Markup Language RDML The file is stored as a compressed file using the PKZIP archive format Export formats and The export formats can be used in combination with the ViiA 7 Software application the ViiA 7 programming interface API to integrate the ViiA 7 System into a laboratory Software API information management system LIMS workflow Applied Biosystems ViiA 7 Real Time PCR System User Guide 199 Calibration Maintenance Networking and Security E Appendix E ViiA 7 Software Reference Export formats and file specifications ViiA 7 export format File structure 200 The ViiA 7 Software can export setup and results data from experiment files eds to tab delimited text files txt in a native ViiA 7 System export format Data exported in the ViiA 7 export format can be opened by common spreadsheet applications such as Microsoft Excel or imported by laboratory information management syst
322. y Appendix D Parts and Materials Calibration and verification kits 96 well sample block kits ViiA 7 System consumable Part Storage number C 96 Well Spectral Calibration Plate with FAM Dye 4432327 15 to 25 96 Well Spectral Calibration Plate with VIC Dye 4432334 96 Well Spectral Calibration Plate with ROX Dye 4432340 96 Well Spectral Calibration Plate with SYBR Green Dye 4432346 96 Well Spectral Calibration Plate with TAMRA Dye 4432352 96 Well Spectral Calibration Plate with NED Dye 4432358 96 Well Region of Interest ROI and Background Plates 4432364 e 96 Well Region of Interest ROI Calibration Plate e 96 Well Background Plate 96 Well Normalization Plates with FAM ROX and VIC ROX 4432370 Dyes e 96 Well Normalization Plate with FAM ROX Dye e 96 Well Normalization Plate with VIC ROX Dye Kit TaqMan RNase P 96 Well Instrument Verification Plate 4432382 e TaqMan RNase P 96 Well Instrument Verification Plate 178 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Appendix D Parts and Materials Calibration and verification kits Fast 96 well sample block kits ViiA 7 System consumable Boe ae Fast 96 Well Spectral Calibration Plate with FAM Dye 4432389 15 to 25 Fast 96 Well Spectral Calibration Plate with VIC Dye 4432396 Fast 96 Well Spectra
323. y seated within the fixture f c Use the two alignment pins in the fixture to position the array card correctly d Seal the array card by running the carriage slowly over it Run the carriage over the array card in one direction only Do not apply downward force on the carriage as you move it forward over the card e Remove the sealed array card from the fixture and trim the fill reservoirs from the array card assembly using scissors Trim the foil array card so S that the edge is even with the plastic carrier IMPORTANT Completely remove the fill reservoirs from the array card so that the edge is free of residual plastic The plastic from the fill reservoirs that extends beyond the edge of the card can prevent the array card from seating properly on the sample block and can affect amplification Correct trim Incorrect trim seer T fe e 12 Repeat step 11 to seal the remaining array cards IMPORTANT As you seal the remaining filled array cards store them in a dark place Do not expose the array cards to light until you are ready to use them The dyes in the array cards are photosensitive Prolonged exposure to light can diminish the fluorescence of the dye IMPORTANT If an array card is sealed improperly the card may leak and contaminate the sample block and or it can cause the associated calibration or RNase P experiment to fail Applied Biosystems ViiA 7
324. y the well 100 character string Target Name The name of the target assay assigned to the well 100 character string Task The task assigned to the target in the well UNKNOWN Or NTC Reporter The reporter dye that labels the probe for the target assay 100 character string Quencher The quencher dye that labels the probe for the target assay 100 character string CT The calculated threshold cycle Cy for the target at the specified well Float Ct Mean The average C of the replicate wells for the specified target sample combination Float Ct SD The standard deviation of the average Cy of the replicate wells for the specified target Float Quantity e Unknown wells The calculated quantity for the sample at the well e Standard wells The quantity assigned to the standard at the well Float Quantity Mean e Unknown wells The average quantity of the replicate wells for the target sample e Standard wells The quantity assigned to the replicate wells for the target sample Float Quantity SD The standard deviation of the average quantity of the replicate wells for the target sample Float Automatic Ct Threshold Whether the threshold was determined automatically true or manually false true Or false Ct Threshold The threshold cycle C7 for the sample at the well Float Automatic Ct Whether the baseline w
325. you have received related training Incorrect lifting can cause painful and sometimes permanent back injury Use proper lifting techniques when lifting or moving the instrument At least two people are required to lift the instrument CAUTION PHYSICAL INJURY HAZARD Do not remove the instrument cover There are no components inside the instrument that you can safely service yourself If you suspect a problem contact an Applied Biosystems Service Representative WARNING PHYSICAL INJURY HAZARD The ViiA 7 System and lamp are hot The lamp can become very hot while in use Allow the lamp to cool for 15 minutes and put on protective powder free gloves before handling it Applied Biosystems ViiA 7 Real Time PCR System User Guide 235 Calibration Maintenance Networking and Security F Appendix F Safety Safety alerts CAUTION PHYSICAL INJURY HAZARD During instrument operation the sample block can be heated to 100 C Before performing the following procedure be sure to wait until the sample block reaches room temperature CAUTION PHYSICAL INJURY HAZARD Wear disposable powder free gloves when handling the lamp to prevent burns and to prevent shortening the life of the replacement lamp 236 Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Documentation and Support Related documentation The following related documents are shipped with the Vii
326. ys the correct type of sample block Note The Block Type field displays the type of sample block installed to the ViiA 7 Instrument Perform the following calibrations in the specified order ROI calibration Background calibration Uniformity calibration Dye calibration then Normalization calibration Applied Biosystems ViiA 7 Real Time PCR System User Guide Calibration Maintenance Networking and Security Chapter5 Service 5 Replace the heated cover Replace the heated cover Replace the heated cover in the event of a hardware failure or if you want to change the consumable format of the ViiA 7 Instrument WARNING PHYSICAL INJURY HAZARD During instrument operation the sample block and heated cover can be heated to 100 C Before removing the heated cover be sure to wait until it reaches room temperature Materials required e Safety glasses e Powder free gloves e Heated cover How to handle the heated cover To prevent damaging or contaminating the heated cover handle the assembly as shown below After the assembly has been removed from the ViiA 7 Instrument place the heated cover on a clean dry surface or in its shipping container Replace the heated cover Note Confirm that the replacement heated cover supports the consumable format that you want to use Some heated covers support more than one consumable type 1 Power off and unplug the ViiA 7 System then allow it to cool for 15 minut

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Applied Biosystems ViiA™ 7 Real-Time PCR System User Guide