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QuantStudio 3D Digital PCR System User Guide (Pub. No

1. INSTRUMENT NAME IMPORTANT The instrument name is an alphanumeric string used to identify the QuantStudio 3D Instrument on the network The instrument name Can be up to 16 characters long Must consist of alphabetic and numeric characters Can contain hyphens but not as the first or last characters in the name Cannot include spaces or special characters lt gt 1 4 Touch when done to save the setting then touch again to return to the Main Menu Set the date and Note If connected to a network the QuantStudio 3D Instrument can automatically time acquires the date and time settings via Network Time Protocol NTP 1 From the Main Menu of the touchscreen touch then touch Instrument Settings 2 In the Instrument Settings screen touch Date Time 3 Set the date time and time zone a Touch the Time Zone field then select the correct continent and region from the lists b Touch the Date field enter the current date then touch Enter c Touch the Time field enter the appropriate time units then touch Enter DATE TIME US Pacific New 2013 05 07 11 53 16 74 QuantStudio 3D Digital PCR System User Guide 4 Maintenance Returning your QuantStudio 3D Instrument for service Touch when done to save the setting then touch again to return to the Main Menu Returning your QuantStudio 3D Instrument for service Before returning your QuantStud
2. CAUTION Cleaning and Decontamination Use only the cleaning and decontamination methods specified in the manufacturer s user documentation It is the responsibility of the operator or other responsible person to ensure the following requirements are met No decontamination or cleaning agents are used that could cause a HAZARD as a result of a reaction with parts of the equipment or with material contained in the equipment The instrument is properly decontaminated a if hazardous material is spilled onto or into the equipment and or b prior to having the instrument serviced at your facility or sending the instrument for repair maintenance trade in disposal or termination of a loan decontamination forms may be requested from customer service Before using any cleaning or decontamination methods except those recommended by the manufacturer users should confirm with the manufacturer that the proposed method will not damage the equipment Safety and electromagnetic compatibility EMC standards Safety EMC 102 The instrument design and manufacture complies with the standards and requirements for safety and electromagnetic compatibility as noted in the following table Reference Description EU Directive European Union Low Voltage Directive 2006 95 EC CSA C22 2 No Safety requirements for electrical equipment for measurement 61010 1 control and laboratory use Part 1 General requirem
3. 0 c ccc eee eee eee eee 91 General use materials and consumables 0c cece cece e eee 92 How to order from the Life Technologies website You can order materials and accessories from Life Technologies by ordering directly from the Life Technologies online store Note Product availability and pricing may vary according to your region or country Online ordering through Life Technologies is not available in all countries Contact your local Life Technologies representative for help To order through the website we recommend the following browsers and Adobe Reader versions to use the Life Technologies website ane i grace Apple Mozilla Adobe perating system gt Explorer Safari Firefox Reader Microsoft Windows v6 x or later v2 0 4 or v2 x or later v4 0 or later later Apple n a IMPORTANT Confirm that cookies and Javascript are turned on for the website to function correctly e To order assays and reagents a Go to www lifetechnologies com b Under I Want to Buy select the product of interest e To order instrument parts and accessories a Go to www lifetechnologies com b Click Parts and accessories c Select the desired components the complete the order as instructed 90 QuantStudio 3D Digital PCR System User Guide Parts and Materials Kits consumables and accessories Kits consumables and accessories P
4. 2 Confirm that you have received the following materials required for the installation of the QuantStudio 3D Instrument e QuantStudio 3D Digital PCR Instrument e QuantStudio 3D Digital PCR Chip Carrier for Dual Flat Block GeneAmp PCR System 9700 and QuantStudio 3D Digital PCR Thermal Pad for Dual Flat Block GeneAmp PCR System 9700 e QuantStudio 3D Digital PCR 20K Chip 12 pack e GeneAmp PCR System 9700 Base Module e QuantStudio 3D Tilt Base for Dual Flat Block GeneAmp PCR System 9700 e QuantStudio 3D Digital PCR Chip Loader e GeneAmp PCR System 9700 Dual Flat Block Module e QuantStudio 3D Digital PCR Master Mix If you have not received one or more of the items listed above contact Life Technologies for a replacement QuantStudio 3D Digital PCR System User Guide 15 Getting Started C Prepare the QuantStudio 3D Digital PCR System for use 3 If you plan to connect your instrument to a network confirm that you have ordered and received either the e Wired network Category 6 Ethernet cable e Wireless network SparkLAN WUBR 170GN 802 11n Single Band Wireless Dongle IMPORTANT Network setup is not required for instrument installation If necessary you can connect your instrument to a network at a later time after the installation 4 Confirm that all chemistry kits and accessories have been stored at the correct temperature TM 5 Unpack and install the QuantStudio 3D Instrum
5. IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply WARNING HAZARDOUS WASTE from instruments Waste produced by the instrument is potentially hazardous Follow the guidelines noted in the preceding General Chemical Handling warning QuantStudio 3D Digital PCR System User Guide 103 Appendix E Safety Biological hazard safety Biological hazard safety 104 WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards 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 All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Safety equipment also may include items for personal protection such as gloves coats gowns shoe covers boots respirators face shields safety glasses or goggles Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially biohazardous materials Follow all applicable local state provincial and or national regulations The following references provide general guidelines when handling biological samples in
6. Total volume 32 4 324 0 11 Volumes include 20 excess to compensate for volume oss from pipetting 5 Using a permanent marker label a 0 5 or 1 5 mL reaction tube for each sample that you intend to run 6 Vortex then briefly centrifuge the DNA samples QuantStudio 3D Digital PCR System User Guide 31 Prepare Samples and Load Reactions Load the Digital PCR 20K Chips 7 Transfer 32 4 uL of PCR reaction mix to each labeled reaction tube 8 Transfer 3 6 uL of each sample diluted to the appropriate concentration to the corresponding reaction tube Mix well by gently pipetting up and down after each transfer or gently vortex on low medium speed 9 Cap the reaction tubes then briefly centrifuge them and immediately proceed to load the Digital PCR 20K Chips IMPORTANT For optimal results load the Digital PCR 20K Chips as soon as possible after setting up the reactions If necessary place the reactions on ice until you are ready to use them If placed on ice warm the samples to room temperature prior to loading Load the Digital PCR 20K Chips Guidelines for loading and sealing Digital PCR 20K Chips 32 TM After preparing your PCR reactions you can load them into QuantStudio 3D Digital PCR 20K Chips manually or automatically using a QuantStudio 3D Digital PCR Chip Loader To load and seal Digital PCR 20K Chips e Automatically See Load the chips using the Chip Loader
7. Touch then touch Maintenance amp Service c Inthe Maintenance amp Service screen touch Software Update to view the software version number to confirm that the firmware has been upgraded 7 When you are finished touch Cancel then touch to return to the Main Menu View the instrument log TM You can use the QuantStudio 3D Digital PCR Instrument touchscreen to view and export a log that summarizes the past 6 months of instrument activity For each event the log provides a description of the activity and the date time when it occurred Note The information conveyed in the instrument log is used primarily by Life Technologies support as a resource for troubleshooting instrument errors If the QuantStudio 3D Instrument encounters an error during operation the log is written to the experiment eds file which can be exported and sent to Life Technologies If QuantStudio 3D Digital PCR System User Guide 77 Maintenance View the instrument log the instrument does not generate a file then you can export the instrument log for distribution as explained below View the log 1 From the Main Menu of the touchscreen touch then touch Maintenance amp Service 2 In the Maintenance amp Service screen touch View Log 3 In the Instrument Log screen review the records of interest 4 Optional Export the system log to a USB drive a Plug the drive into the USB port on the front of the QuantStudio 3D
8. directly from the service section of the Life Technologies website After obtaining a firmware update transfer the update to the QuantStudio 3D Digital PCR Instrument using a USB drive 76 QuantStudio 3D Digital PCR System User Guide Maintenance View the instrument log Update the 1 Download the firmware update firmware a Go to www lifetechnologies com support software b In the Software Downloads page select Applied Biosystems QuantStudio 3D Digital PCR System Software from the menu c In the Software Downloads page for your QuantStudio 3D Digital PCR Instrument click Updates Patches d Download the QuantStudio 3D Instrument firmware to a USB drive 2 Plug the drive into the USB port on the front of the QuantStudio 3D Instrument 3 From the Main Menu of the touchscreen touch then touch Maintenance amp Service 4 In the Maintenance amp Service screen touch Software Update then wait while the QuantStudio 3D Instrument scans the USB drive for the firmware update 5 In the Software Upgrade screen touch OK to proceed with the update Allow the QuantStudio 3D Instrument to complete the upgrade IMPORTANT Do not remove the USB drive from the QuantStudio 3D Instrument until after the update is complete 6 After the upgrade is complete and the QuantStudio 3D Instrument restarts confirm the success of the firmware update a Unplug the USB drive from the instrument b
9. on page 33 e Manually See Load chips manually on page 41 To ensure the proper function of your loaded Digital PCR 20K Chip handle them according to the following guidelines e Always wear powder free gloves when loading and sealing Digital PCR 20K Chips IMPORTANT Never handle Digital PCR 20K Chips or Chip Case Lids without gloves Oils from your hands can contaminate the components and interfere with thermal cycling and imaging e Handle the Digital PCR 20K Chips and Chip Case Lids as follows Hold the Digital PCR 20K Chips and Chip Case Lids by gently gripping them by their sides Do not touch the surface of the Digital PCR 20K Chips If you accidentally touch the surface of a chip discard it Note Debris or excess sample on the Chip Case Lid can fluoresce and interfere with thermal uniformity 9E000E0X O a QuantStudio 3D Digital PCR System User Guide Prepare Samples and Load Reactions ye Load the Digital PCR 20K Chips If necessary use a permanent pen or marker to label the back of each loaded Chip Case to aid in sample tracking Load each Digital PCR 20K Chip within 2 hours after opening it Load Digital PCR 20K Chips in alphanumeric order according to Digital PCR 20K Chip serial number to avoid data entry errors Seal each Digital PCR 20K Chip immediately after loading it to avoid evaporation Load and seal Digital PCR 20K Chips in batches of 24 or less the
10. 0 cece eee eee ees 59 Analyzethe chips 2 5 occ25 02 cee one gcas Re awe Rate peewee eee eee eee 60 Materials required 0 0 cece eee eee eee 60 Guidelines for high throughput analysis 00 cee eee eee eee eee 60 Image the Digital PCR 20K Chips 2 0 00 cece eee 60 Analyzing the data daneen aneus Head e e Bee hone radia Meee eee 63 AOU TMG analysis essisd x bent cet pnn ded sd ag ee Gene aaa ac cone ar wll ee 63 About the results screen 22 e ee eee ee eee eens 64 About data quality flags 0 0 2 c eee eee eee 65 About the data files o 23 ccha ck at keneren tai eenn ee eee wel aed a 65 Transfer the resultS 2 2 0 eee eee eee eee eee 66 Using the QuantStudio 3D AnalysisSuite Software 2 cece eee ee eee 67 Computer requirements 2 00 c cece eee eee eee ees 67 Access the software from a cloud account 0 eee ee ee eee eee eee 68 Obtaining information from the Help system 0002 c cece eee eee eee 68 CHAPTER 5 Troubleshooting cccceecee eee ee eeeeeeeeeeeees 69 Troubleshooting chip images using the Chip View 220 eee eee ee eee eee 69 QuantStudio 3D Digital PCR System User Guide Contents APPENDIXA Maintenance 0ccccee eee ee eee eee eee ee eens 72 Cleaning the chip tray and sample block 000 c eee eee eee eee eee 72 Matertals required 00 45 2chtiicy beer tian hee E ewes goer
11. 3D Instrument e Subnet mask if applicable e Gateway address if applicable e Internet domain name e Domain Name Server address es e Windows domain if applicable e WINS server address es if applicable If connecting your instrument to a wireless network obtain the following information for the local wireless hotspot e Wireless local area hotspot name e Security policy requirement for the wireless hotspot WPA WEP or WPA2 e Network credentials for the wireless hotspot password and user account if necessary QuantStudio 3D Digital PCR System User Guide 81 Appendix B Networking Connect the instrument to the network Connect the QuantStudio 3D Instrument to a network 82 1 Prepare the physical connection to the network Connection Action Wired Power on the QuantStudio 3D Instrument Connect one end of a standard category 6 Ethernet cable to the RJ 45 port on the back panel of the instrument and the other end to an open network port Wireless 3 Turn the QuantStudio 3D Instrument over then use your Turn the instrument over then uncap the wireless dongle and If required for network setup record the wireless dongle s MAC address which is located on the underside of the device Power off and unplug the QuantStudio 3D Instrument fingers to carefully pry open the rear panel IMPORTANT The rear panel is secured to the instrument by three pegs that require signifi
12. Instrument b In the Instrument Log screen touch Export then wait for the instrument to export the log to a text file on the drive INSTRUMENT LOG x 02_11 52 08_PDT eds to USB target USB 11 43 08 169 Warning Failed to change state Storage M ounted from Error to True in trigger MountStorage I iS User NoStorageDevice gt No storage device is availa 11 43 08 332 Warning Failed to transfer XO40098N_2013 05 02_11 52 08_PDT eds IS User NoStorageDevice gt No storage device is available 11 43 08 342 State Experiment Exporting False Export 5 When you are finished touch X then touch to return to the Main Menu 78 QuantStudio 3D Digital PCR System User Guide Networking IMPORTANT This document does not provide adequate detail to integrate the QuantStudio 3D Instrument into all possible network architectures Because your laboratory network can contain advanced features such as firewalls Life Technologies recommends that you consult a network administrator before connecting your instrument Networking overview About the network port and wireless adapter TM fl After installing the QuantStudio 3D Digital PCR Instrument and the QuantStudio 3D AnalysisSuite Software you can connect the QuantStudio 3D Instrument to a local area network to enhance its functionality When connected to a network the QuantStudio 3D Instrument can upload data directly to the Qua
13. QuantStudio 3D Digital PCR Instrument optics collect the residual fluorescence emitted from the wells of the Digital PCR 20K Chip The resulting image collected by the device consists of light corresponding to the range of emission wavelengths for the filter in use 3 Interpretation The QuantStudio 3D Digital PCR Instrument assembles a digital representation of the residual fluorescence collected over a fixed time interval TM After a run the QuantStudio 3D Digital PCR Instrument determines the location and intensity of the fluorescent signals in each image the dye associated with each fluorescent signal and the significance of the signal TM The QuantStudio 3D Digital PCR System features a filter set that is optimized for the FAM VIC and ROX dyes available from Life Technologies QuantStudio 3D Digital PCR System User Guide 11 Getting Started CA Operating the instrument Operating the instrument TM Compatible The QuantStudio 3D Digital PCR System can be used to perform experiments using reagent kits and compatible reagent kits and assays sold by Life Technologies For specific information consumables on a reagent kit or assay refer to the documentation accompanying the product or consult the Life Technologies website Operational The following figure shows the workflow for performing a single experiment on the workflow QuantStudio 3D Digital PCR System The procedures for sample and or consumable
14. Sample Block Module includes a heated cover that is manually positioned over the sample block prior to the PCR run The cover applies pressure to the Digital PCR 20K Chips to seat them firmly and precisely on the sample block ensuring proper heat transfer QuantStudio 3D Digital PCR System User Guide 49 5 Perform the PCR STT Choosing a thermal cycler Prepare the Before running each batch of Digital PCR 20K Chips perform the following to ensure GeneAm p PCR efficient thermal cycling System 97700 1 Confirm that the following parts are installed or available for use TM a Confirm that clean QuantStudio 3D Digital PCR Thermal Pads are available for use IMPORTANT If a QuantStudio Thermal Pad becomes dirty clean the pad using a lint free lab wipe sprayed with isopropanol then let it dry Fluids debris and other contaminants that adhere to the pad can transfer to the Digital PCR 20K Chips during thermal cycling and fluoresce during imaging IMPORTANT Use only QuantStudio Thermal Pads that are approved for use with Digital PCR 20K Chips Thermal compression pads from other PCR applications may not apply uniform pressure or heat distribution to all chips on the sample block b Confirm that the QuantStudio 3D Tilt Base for Dual Flat Block GeneAmp PCR System 9700 is installed beneath the thermal cycler IMPORTANT The Tilt Base inclines the GeneAmp PCR System 9700 sample block by 11 the angle re
15. Select User Name screen displays a list of names of all users who have been added to the instrument displays in a 4 x 5 matrix 08 00 AM 4 25 13 GeneAmp PCR System 9700 Version 3 12 Name tc001 User lt lt pe gt gt Run Create Edit Util User Press New F2 to add a new name to the list Select User Name lt lt pe gt gt adrian doug peter Accept New Edit Delete Cancel In the User Name field enter an alphanumeric name up to six characters in length then press Accept F1 to accept a name abcdefghi jklmnopqrj stuvwxyz User Name op t t Use ENTER key to select a character Backsp Cancel The name you add or the name you select from a list of existing user names becomes the current user name All new methods that you create are stored by default under the current user name Configure the Security Code screen according to your needs User Name hank PIN number None Protection Unlocked Press PIN to create a Then you set protection to Locked to prevent methods from being overwritten or deleted Accept Name PIN QuantStudio 3D Digital PCR System User Guide Getting Started E 7 Connect the QuantStudio 3D Instrument to a network i If you Then do not want to protect your methods press Accept F1 again want to protect your methods see the GeneAmp PCR System 9700 User Manual Pub no 4303481 for instru
16. based or SAMBA file server over the Server Message Block SMB protocol e Date and time synchronization via Network Time Protocol NTP TM TM The wireless dongle sold with the QuantStudio 3D Digital PCR Instrument can be installed to an internal USB port behind the rear panel to enable wireless network QuantStudio 3D Digital PCR System User Guide 79 Appendix B Networking Connect the instrument to the network connectivity See the product documentation accompanying the adapter for the network requirements specifications and product details Networking e We recommend that you consult a network administrator before connecting the guidelines and QuantStudio 3D Instrument to your laboratory network best practices e We recommend that you name the QuantStudio 3D Instrument uniquely While not critical to network function unique instrument names can aid with sample tracking and instrument troubleshooting Note The serial number of the QuantStudio 3D Instrument is the default instrument name The QuantStudio 3D Instrument does not test the uniqueness of the instrument name when it is set e Name QuantStudio 3D Instruments using numbers and letters The instrument name can include hyphens but not as the first or last character Connect the instrument to the network Network setup Start workflow v If dynamic network configuration via DHCP is not available or if you are uncertain collect the required net
17. connection selected no connection available e amp Wireless connection selected connected to network e amp Wireless connection selected no connection available Data destination icon Represents the selected data destination e Cloud destination selected e CP USB drive destination selected File name Name of the experiment eds file created for the imaged Digital PCR 20K Chip Note After the analysis the instrument copies the file to the selected destination and stores the file locally Scroll buttons Allow you to display the results of previously imaged Digital PCR 20K Chips when running them ina series QuantStudio 3D Digital PCR System User Guide About data quality flags About the data files Analyze the Prepared Chips EE Analyzing the data CS3 eae me Data quality flags generated and displayed by the software are a measure of the overall chip quality based on well quality thresholds and other data analysis characteristics Note For more information on quality assessment in the software see the QuantStudio 3D AnalysisSuite Software Help In order from high quality to low quality e P green Review of the analysis results is not required This flag is shown if the data meets all quality thresholds e M yellow Review of the analysis results is suggested This flag is shown if the Instrument cannot clearly identify the population of unamplified wells sigma
18. for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document Symbols on this instrument 98 Symbols may be found on the instrument to warn against potential hazards or convey important safety information In this document the hazard symbol is used along with one of the following user attention words e CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices e WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury e DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury Symbol English Francais A Caution risk of danger Attention risque de danger Consult the manual for further safety Consulter le manuel pour d autres information renseignements de s curit Ultraviolet light Rayonnement ultraviolet Protective conductor terminal main Born
19. laboratory environment U S Department of Health and Human Services Biosafety in Microbiological and Biomedical Laboratories BMBL 5th Edition HHS Publication No CDC 21 1112 Revised December 2009 found at www cdc gov biosafety publications bmb15 BMBL pdf World Health Organization Laboratory Biosafety Manual 3rd Edition WHO CDS CSR LYO 2004 11 found at www who int csr resources publications biosafety Biosafety7 pdf QuantStudio 3D Digital PCR System User Guide Obtaining SDSs Documentation and support Safety Data Sheets SDSs are available from www lifetechnologies com suppott Note For the SDSs of chemicals not distributed by Life Technologies Corporation contact the chemical manufacturer Obtaining Certificates of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Obtaining support For the latest services and support information for all locations go to www lifetechnologies com support At the website you can Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support techsupport lifetech com Searc
20. loading them IMPORTANT Store the prepared Digital PCR 20K Chips in a clean dry dark location until you are ready to load them onto the GeneAmp PCR System 9700 48 QuantStudio 3D Digital PCR System User Guide a A YA A 1 LIN IEY EnS gt N 7 wW Perform the PCR Em Choosing a thermal cycler 6 6 6 eect eee 49 m Thermal cycle the Digital PCR 20K Chips 0 0 c cece eee ees 52 Choosing a thermal cycler About the Dual Flat Block GeneAmp PCR System 9700 The Dual Flat Block GeneAmp PCR System 9700 is the only thermal cycler that can be used to run prepared QuantStudio 3D Digital PCR 20K Chip IMPORTANT Due to the physical characteristics of the Digital PCR 20K Chips no other thermal cycler can be substituted for the GeneAmp PCR System 9700 The GeneAmp PCR System 9700 is an automated thermal cycler designed for the amplification of nucleic acids using the Polymerase Chain Reaction PCR process The user interface consists of a control panel with a full numeric keypad soft keys and a graphical display screen that shows the time and temperature profile for each run The Dual Flat Block Sample Module is an interchangeable sample block that attaches to the top of the GeneAmp PCR System 9700 Base Module The module features two flat aluminum sample blocks that are designed to perform thermocycling of up to 24 sealed Digital PCR 20K Chips simultaneously The Dual Flat
21. maximum number that can be loaded onto the GeneAmp PCR System 9700 If you do not intend to load an opened Digital PCR 20K Chip immediately cover the chip using the aluminum plate found within the packaging to prevent contamination When not in use store the Chip Sealant within its original protective package to prevent the sealant in the syringe tip from curing The Chip Sealant can be stored with the syringe tip attached Use all of the QuantStudio 12K Flex OpenArray Immersion Fluid within 60 minutes of uncapping the syringe Once a syringe is opened you cannot reattach the cap for later use Thermal cycle Digital PCR 20K Chips within 2 hours after loading them Load the chips The following procedure explains how to load QuantStudio 3D Digital PCR 20K using the Chip Chips automatically using a QuantStudio 3D Digital PCR Chip Loader This Loader procedure assumes that you have prepared your PCR reactions as explained in Prepare the reaction mix and samples on page 31 and are ready to load them onto Digital PCR 20K Chips Materials required TM The following materials are required to load and seal QuantStudio 3D Digital PCR 20K Chips for use on the QuantStudio 3D Digital PCR System Prepared digital PCR reactions from the previous section QuantStudio 3D Digital PCR 20K Chips QuantStudio 3D Digital PCR Chip Case Lids QuantStudio 3D Digital PCR Sample Loading Blades QuantStudio 12K FlexOpenArray
22. one small air bubble lt 2 3 mm in diameter is acceptable 9E000E0X e iO b Using a lint free laboratory wipe remove any excess Immersion Fluid from the Chip Case to ensure optimal imaging on the QuantStudio 3D Instrument 7 Seal the Chip Case using Chip Sealant a Insert the syringe tip into the fill port of the sealed Chip Case then carefully fill the port with Chip Sealant ensuring that the fluid touches the walls of the hole When full finish the seal by lifting the syringe tip and add a small amount of sealant over the top of the port IMPORTANT Apply the Chip Sealant to the fill port only Do not apply the sealant to the Chip Case Lid window IMPORTANT When not in use store the Chip Sealant inside its original protective package and with the syringe tip attached to prevent the sealant in the tip from curing 9 000 0X QuantStudio 3D Digital PCR System User Guide 39 40 Prepare Samples and Load Reactions Load the Digital PCR 20K Chips b Insert the Digital PCR 20K Chip assembly into the UV Curing Station on the Chip Loader Push the chip into the station until the ultraviolet light illuminates IMPORTANT Make sure that the chip is oriented so that the fill port is directly beneath the beam of ultraviolet light IMPORTANT Do not allow the Digital PCR 20K Chip to contact the roof of the UV Curing Station ee 9E000E0X Q G When the light powers off a
23. preparation and result analysis can vary depending on the specific experiment that you are performing Start v Set up the PCR reaction by mixing sample master mix and assay s v Load the PCR reaction onto a QuantStudio 3D Digital PCR 20K Chip apply a lid load the assembly with immersion fluid then seal the loading port v Perform the PCR using the Dual Flat Block GeneAmp PCR System 9700 v Read the Digital PCR 20K Chip using the QuantStudio 3D Digital PCR Instrument v Review the results on the QuantStudio 3D Instrument touchscreen v Store or discard the Digital PCR 20K Chip v Analyze the data using the QuantStudio 3D AnalysisSuite Software v Finish TM TM Note Refer to the QuantStudio 3D AnalysisSuite Software User Guide Pub no MAN0008161 for detailed information on data analysis 12 QuantStudio 3D Digital PCR System User Guide Getting Started Prepare the QuantStudio 3D Digital PCR System for use Rage Prepare the QuantStudio 3D Digital PCR System for use About the installation Materials required Before you begin Plan the laboratory layout TM You can install the QuantStudio 3D Digital PCR System within one hour The installation requires your participation at all times so plan to spend most of your time working with the instrument TM Note The time required to install the QuantStudio 3D System can vary depending on the type of networking sol
24. primary top and secondary bottom WINS servers IMPORTANT WINS may be required if you specify a file server as a data destination Note During operation the instrument attempts to resolve WINS names to IP addresses using the server specified in the top most field If unavailable the instrument uses the server specified in the bottom most field NETWORK SETUP f When you are finished configuring your network settings touch OK The touchscreen displays Network Complete when the instrument successfully connects to the network NETWORK COMPLETE Vv You are now connected to Ethernet 86 QuantStudio 3D Digital PCR System User Guide ree Appendix B Networking Connect the instrument to the network 7 In the Network Complete screen touch OK then touch to return to the Main Menu After the instrument has successfully connected to the network the Network Setup screen displays the details of the network connection 4 Wired Connected DHCP Ta 10 10 10 10 255 255 252 0 10 10 10 10 Note If your wireless hotspot performs MAC filtering you can obtain the Media Access Control MAC address of the QuantStudio 3D Instrument on the label of the wireless dongle that you installed in step 1 Create a network The following procedure allows you to create network data destinations to receive destination to experiment eds files generated by the QuantStudio 3D Instrument You can cre
25. separate but contiguous room make sure that air flows toward the amplified DNA area In addition it is helpful if there is a separate exit from the post PCR work area that does not exit into the pre PCR work area Note If possible the station for loading Digital PCR 20K Chips should be physically separated from the PCR area away from the GeneAmp PCR System 9700 and the QuantStudio 3D Instrument Choose the instrument locations Placement of the QuantStudio 3D Digital PCR Instrument and GeneAmp PCR System 9700 can greatly affect their performance For both instruments select installation locations that satisfy all of their specific environmental requirements To prevent vibration place the instruments on a solid stable level surface that allows free airflow overhead and around the sides and backs To ensure optimal heat dissipation keep all instrument ventilation slots free of obstructions Instrument For environmental requirements see QuantStudio 3D Digital PCR Environmental requirements on page 95 Instrument GeneAmp PCR System 9700 GeneAmp PCR System 9700 Base Module User Manual Pub no 4303481 14 QuantStudio 3D Digital PCR System User Guide Choose additional protective devices Install the QuantStudio 3D Digital PCR System Getting Started Prepare the QuantStudio 3D Digital PCR System for use SAD We recommend several electrical devices to protect the systems in environments with large voltage
26. separation lt 5 Distribution of unamplified wells on the chip is not uniform uniformity assessment lt 0 99 Sample concentration is outside the optimal range 200 2000 copies uL currently defined by the Life Technologies chip quality metrics e P red Review of the analysis results is strongly suggested If necessary reimage the Digital PCR 20K Chip or rerun the sample This flag is shown if the total number of filled wells is lt 5000 or the percentage of low quality wells those with well quality lt 0 5 is gt 15 For each QuantStudio 3D Digital PCR 20K Chip it reads the QuantStudio 3D Digital PCR Instrument stores the processed imaging data and the results of the preliminary analysis to a single experiment eds file The instrument names each experiment file uniquely by combining the ID of the chip and the date time of the run according to the following formula BO498N_ 130531 195817 eds Chip ID The label applied to the Date stamp The date at which the QuantStudio 3D Digital PCR Chip Case Digital PCR 20K Chip was run expressed Lid that uniquely identifies the Digital in the yymmda format where yyis the PCR 20K Chip to which it is applied last two digits of the year mm is the numerical order of the month and ddis the day of the run Time stamp The time at which the T y0300095 Chip ID Digital PCR 20K Chip was run expressed ee in the AAmmss format where Ahis
27. within 2 hours after loading them IMPORTANT Store the prepared Digital PCR 20K Chips in a clean dry dark location until you are ready to load them onto the GeneAmp PCR System 9700 TM Load chips The following procedure explains how to load QuantStudio 3D Digital PCR 20K manually Chips manually using a UV Stylus and UV Curing Stylus Stand It can be used as an alternative method for loading chips if a QuantStudio 3D Digital PCR Chip Loader is unavailable This procedure assumes that you have prepared your PCR reactions as explained in Prepare the reaction mix and samples on page 31 and are ready to load them onto Digital PCR 20K Chips Materials required The following materials are required to load and seal QuantStudio 3D Digital PCR 20K Chips for use on the QuantStudio 3D Digital PCR System e Prepared digital PCR reactions from the previous section e QuantStudio 3D Digital PCR 20K Chips e QuantStudio 3D Digital PCR Chip Case Lids e QuantStudio 3D Digital PCR Sample Loading Blades e QuantStudio e QuantStudio 12K Flex OpenArray Immersion Fluid Tip e UV Activated Chip Sealant Syringe e UV Curing Stylus for Chip Sealant e UV Curing Stylus Stand e Isopropanol 12K Flex OpenArray Immersion Fluid e Gloves powder free nitrile e Heated block capable of maintaining 40 1 C e Scissors e Laboratory grade lint free wipes e Microcentrifuge e Pipettes and tips P10 to P1000 e Vortexer QuantSt
28. Immersion Fluid QuantStudio 12K FlexOpenArray Immersion Fluid Tip QuantStudio 3D Digital PCR Chip Loader UV Activated Chip Sealant Syringe Isopropanol Gloves powder free nitrile Scissors Laboratory grade lint free wipes Microcentrifuge Pipettes and tips P10 to P1000 Vortexer QuantStudio 3D Digital PCR System User Guide 33 Gi Prepare Samples and Load Reactions 2 Load the Digital PCR 20K Chips QuantStudio 3D Digital PCR Chip Loader status light The QuantStudio 3D Digital PCR Chip Loader features an LED at the top of the Chip Loader that indicates its status The status light is illuminated at all times when the Chip Loader is powered on and changes color according to instrument readiness Color Status Blue solid The Chip Loader is booting White flashing The chip nest is not at temperature or the lever arm is not fully open Green solid The Chip Loader is ready to load a Digital PCR 20K Chip Green flashing The Chip Loader is loading a Digital PCR 20K Chip Red flashing The Chip Loader has encountered an error Recovering from a Chip Loader error Perform the following steps if the QuantStudio 3D Digital PCR Chip Loader status light flashes red indicating that it has encountered an error 1 Power off the Chip Loader wait 30 seconds then power it on 2 Ifthe status light is still flashing red press the load button and record the sequence of colors dis
29. PORTANT Before applying the Chip Case Lid make certain that it is correctly oriented to the Digital PCR 20K Chip assembly e Briefly vortex and centrifuge the prepared digital PCR reaction master mix assay and template then carefully transfer 14 5 uL of the solution into the sample loading port of the Sample Loading Blade If the reactions were placed on ice allow them to warm prior to loading While filling the Sample Loading Blade e Hold the pipet so that the tip meets the sample loading port at a sharp angle and so it does not deflect either blade forward or rear e Do not depress the pipette to the second stop Doing so increases the chance of introducing an air bubble to the Sample Loading Blade e Avoid creating air bubbles ry QuantStudio 3D Digital PCR System User Guide 37 5 L 38 Prepare Samples and Load Reactions Load the Digital PCR 20K Chips f Press the black loading button on the Chip Loader to load the Digital PCR 20K Chip The status light flashes green during the loading sequence and displays solid green when finished After the Chip Loader loads the Digital PCR 20K Chip immediately add 10 15 drops of Immersion Fluid directly onto the chip so that the fluid covers the entire surface If any fluid is present on the edges of the case that will contact the lid remove it with a lint free wipe that has been sprayed with isopropanol IMPORTANT Pipet the Immersion Fluid o
30. QuantStudio 3D Digital PCR 20K Chip leaks during thermal cycling or during a run perform the following procedure to eliminate any residual contaminants from the QuantStudio 3D Instrument chip tray or the GeneAmp PCR System 9700 Materials e Cotton or nylon swabs and lint free cloths required e Deionized water e Isopropanol e Pipette 100 uL with pipette tips e Powder free gloves e Safety glasses e Tissue lint free 72 QuantStudio 3D Digital PCR System User Guide Maintenance Configure the instrument settings Clean the chip tray CAUTION PHYSICAL INJURY HAZARD Do not remove the covers to the or sample block QuantStudio 3D Instrument or GeneAmp PCR System 9700 There are no components inside the instruments that you can safely service yourself If you suspect a problem contact a Life Technologies Service Representative AN 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 4 CAUTION Before using a recommended decontamination method other than that here contact Life Technologies that the proposed method will not damage the equipment IMPORTANT Wear powder free gloves when you perform this procedure 1 Power off and unplug the QuantStudio 3D Instrument or GeneAmp PCR System 9700 2 Open the QuantStudio 3D Instrumen
31. SB drive icon C when the QuantStudio 3D Instrument has mounted the USB drive 3 Open the chip tray and load the Digital PCR 20K Chip face up into the bay Confirm that the Digital PCR 20K Chip is correctly aligned within the chip tray then close it Note The chip tray is keyed such that the Digital PCR 20K Chip will only fit in one orientation Note The chip tray is fully closed when it is flush with the front of the instrument 1 Orient the chip ID and fill port toward 2 Confirm that the chip window is clean the front of the instrument and correctly aligned to the chip The touchscreen illuminates the Start Run button and displays the chip ID when it is ready to perform the run If the QuantStudio 3D Instrument cannot read the ID number the touchscreen displays the instrument name followed by the time and date of the run Start Run B0498N Start Run QuantStudio 3D Digital PCR System User Guide 61 SF Di 62 Analyze the Prepared Chips Analyze the chips 4 In the instrument touchscreen touch Start Run then wait for the QuantStudio 3D Instrument to image the chip IMPORTANT Do not open the chip tray or remove the USB drive if present while the instrument displays the countdown screen Doing so will invalidate the image data and require you to repeat the run Processing Chip 0 21 emm While the instrument images the chip the touchscreen counts down the time remainin
32. Syringe 4 Lint free wipe Cap remove QuantStudio 12K Flex OpenArray Immersion Fluid Tip push to attach 4 Remove the following consumables from their packaging and place them on a clean dry lint free surface e QuantStudio 3D Digital PCR Chip Case Lid e QuantStudio 3D Digital PCR Sample Loading Blade QuantStudio 3D Digital PCR System User Guide 43 Prepare Samples and Load Reactions Load the Digital PCR 20K Chips 5 Open the QuantStudio 3D Digital PCR 20K Chip package gently grasp the chip by its sides then place it face up on the heated block preheated to 40 C IMPORTANT Allow the Digital PCR 20K Chip to sit on the heated block for at least 5 seconds before loading it IMPORTANT Place only one Digital PCR 20K Chip on the heated block at a time Note If desired use a permanent pen or marker to label the back of the Digital PCR 20K Chip Marking the back of the chips will not affect the imaging data Lr 6 Load the PCR reaction onto the Digital PCR 20K Chip a Briefly vortex and centrifuge the prepared digital PCR reaction master mix assay and template then aspirate 14 5 uL of the solution into a P20 pipette If the reactions were placed on ice allow them to warm prior to loading b While holding the Sample Loading Blade by its edges carefully dispense the reaction into the sample loading port After loading gently tap the top of the Sample Loading Blade to
33. USER GUIDE applied biosystems by Kafe technologies QuantStudio 3D Digital PCR System for use with QuantStudio 3D Digital PCR Instrument Dual Flat Block GeneAmp PCR System 9700 QuantStudio 3D Digital PCR Master Mix QuantStudio 3D Digital PCR 20K Chip QuantStudio 3D Digital PCR Chip Loader Catalog Number 4482593 and 4481097 Publication Number MANO007720 Revision A O QUANT STUDIO 3D o technologies For Research Use Only Not for use in diagnostic procedures The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S 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 Important Licensing Information These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses Trademarks The trademarks mentioned herein are the property of Life Technolog
34. al PCR 20K Chips If missing program the GeneAmp PCR System 9700 with the following method See the GeneAmp PCR System 9700 Base Module User Manual Part no 4303481 for more information on programming methods e Protocol Stage Step eee pes Ramp rate Hold INA poiymerase 96 0 10 00 activation Cycling Anneal extend 60 0 2 00 39 cycles Denature 98 0 0 30 Delsult Hold Final extension 60 0 2 00 Hold Storage 10 0 99 59 e Run speed Standard e Reaction volume 20 uL 1 Hold 2 Tmp 39 Cycles 2 Holds 96 0 98 0 Start Method exp 001 QuantStudio 3D Digital PCR System User Guide 51 3 Perform the PCR Thermal cycle the Digital PCR 20K Chips Thermal cycle the Digital PCR 20K Chips Materials The following materials are required to thermal cycle prepared QuantStudio 3D required Digital PCR 20K Chips for use with the QuantStudio 3D Digital PCR System e Prepared QuantStudio 3D Digital PCR 20K Chips from the previous section e GeneAmp PCR System 9700 Base Module e Dual Flat Block Module GeneAmp PCR System 9700 e QuantStudio 3D Digital PCR Chip Carriers e QuantStudio 3D Digital PCR Thermal Pads e QuantStudio 3D Digital PCR Tilt Base e Isopropanol e Gloves powder free nitrile e Laboratory grade lint free wipes Guidelines for To ensure the proper function of your loaded Digital PCR 20K Chips handle the Chip handling Chip Cases according to the followi
35. alculation of the target concentration In this case the sample must first be diluted prior to running the digital PCR experiment TM In a digital PCR experiment performed on an QuantStudio 3D Digital PCR System gDNA samples are diluted down to a limiting quantity such that most individual PCR reactions contain either zero or one target molecule The procedure for determining the optimal dilution for a sample differs depending on whether or not the target copy number per genome is known If the target copy number per genome of your samples is known dilute the samples so that when loaded on a QuantStudio 3D Digital PCR 20K Chip each through hole reaction will contain approximately 0 6 to 1 6 copies of the target sequence For example assuming 3 3 pg copy of a given gene are present per genome and a 865 pL reaction well volume the stock gDNA in a given sample would be diluted down to 600 copies uL or 1 98 ng uL in the final reaction to give 0 6 copies per reaction well How to determine the target copy number per genome To help determine copy number per genome collect the following information 1 If the source or species of the gDNA is known but the genome size of the organism of interest is unknown refer to http www cbs dtu dk databases DOGS index html to determine the size of the genome in question 2 Once the size of the genome is known determine the mass of the genome using the following formula m n 1 096 x 10 g
36. an Assay s for your experiment QuantStudio 3D Digital PCR Master Mix Gloves powder free nitrile Laboratory grade lint free wipes Microcentrifuge Permanent marker or pen Pipettes and tips P10 to P1000 Reaction tubes DNase RNase free non stick or low binding 0 5 mL or 1 5 mL TE Buffer 1X Molecular Biology Grade Vortexer Water DNase free sterile filtered Guidelines for When preparing samples for PCR amplification PCR sample preparation 30 Use a positive displacement pipette or aerosol resistant pipette tips Follow proper pipette dispensing techniques to prevent aerosols Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation Change gloves whenever you suspect that they are contaminated Maintain separate areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Centrifuge tubes before opening Try not to splash or spray PCR samples Keep reactions and components capped as much as possible Clean lab benches and equipment periodically with 10 bleach solution Use DNAZap Solution PN AM9890 QuantStudio 3D Digital PCR System User Guide Prepare the reaction mix and samples Prepare Samples and Load Reactions 5 Prepare the digital PCR react
37. an diminish the fluorescence of the photosensitive dyes in the chips Note If a Digital PCR 20K Chip is sealed improperly the chip may leak and contaminate the sample block If you suspect that a chip has leaked remove the fluid from the sample block using a lint free laboratory wipe sprayed with isopropanol QuantStudio 3D Digital PCR System User Guide a Z 4 Analyze the Prepared Chips 5 lt E 0 4 m Using the QuantStudio 3D Digital PCR Instrument 004 57 M Analyze the chips ease uine nn EE EEEE E RES EE PA NE nee 60 E Analyzing the datas pesetiror ipsia ios ae udana a aE 63 m Using the QuantStudio 3D AnalysisSuite Software 04 67 Using the QuantStudio 3D Digital PCR Instrument TM About imaging and The QuantStudio 3D Digital PCR Instrument performs the image capture and primary analysis subsequent preliminary analysis of QuantStudio 3D Digital PCR 20K Chips following thermal cycling on the GeneAmp PCR System 9700 The QuantStudio 3D Instrument processes Digital PCR 20K Chips individually where each run lasts approximately one minute After reading a chip the instrument performs an initial evaluation of the imaging data collected during the run and displays the results on the instrument touchscreen QuantStudio 3D System data flow For each imaged Digital PCR 20K Chip the QuantStudio 3D Instrument generates and saves a single experiment eds file
38. 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 voltage Power fluctuations can adversely affect the function of the systems Note A power line regulator monitors the input current and adjusts the power supplied to the systems It does not protect against a power surge or failure 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 systems Note A dedicated line and ground between the systems and the building s main electrical service can also prevent problems caused by power fluctuations 1 Plan the organization of your laboratory Identify the locations of all stations in the QuantStudio 3D Digital PCR Instrument workflow including e Reagent and consumable storage e PCR reaction preparation e QuantStudio 3D Digital PCR 20K Chip loading and sealing e Dual Flat Block GeneAmp PCR System 9700 for PCR amplification e QuantStudio 3D Digital PCR Instrument for imaging and analysis Note See Plan the laboratory layout on page 13 for more information on planning the instrument placement
39. antStudio 3D Digital PCR System e Prepared Digital PCR 20K Chip from the previous section e Isopropanol e Gloves powder free nitrile e Laboratory grade lint free wipes Guidelines for For optimal efficiency when running large numbers gt 6 of Digital PCR 20K Chips high throughput follow the guidelines below analysis e Run the Digital PCR 20K Chips in alphanumeric order according to chip ID to help avoid mistakes when entering sample information e If you are running multiple chips you can run the next chip in the series when the touchscreen displays the Analyzing Chip screen Analyzing Chip Analyzing Do not remove USB Data You may insert another chip Image the Digital 1 Set the destination for the imaging data PCR 20K Chips a From the Main Menu of the QuantStudio 3D Instrument touch Destination b In the Destination screen touch the desired destination for the imaging data You can configure the QuantStudio 3D Instrument to upload the imaging data to a USB drive a network file server or the cloud data storage service 4 EDIT DESTINATIONS Test Server Instrument SAMBA Fileshare Life Technologies 60 QuantStudio 3D Digital PCR System User Guide Analyze the Prepared Chips A Analyze the chips T 2 If you choose to use a USB drive as a destination for your imaging data insert the drive into the USB port on the front of the instrument O Heg The touchscreen displays the U
40. art number Description 4485507 QuantStudio 3D Digital PCR 20K Chip Kit 12 pack e QuantStudio 3D Digital PCR 20K Chip 12 e QuantStudio 3D Digital PCR Chip Case Lid 12 e QuantStudio 3D Digital PCR Sample Loading Blade 12 e QuantStudio 12K Flex OpenArray Immersion Fluid 3 e QuantStudio 12K Flex OpenArray Immersion Fluid Tip 3 4482603 QuantStudio 3D Digital PCR 20K Chip Kit and Master Mix e QuantStudio 3D Digital PCR 20K Chip 96 e QuantStudio 3D Digital PCR Chip Case Lid 96 e QuantStudio 3D Digital PCR Sample Loading Blade 96 e QuantStudio 12K Flex OpenArray Immersion Fluid 24 e QuantStudio 12K Flex OpenArray Immersion Fluid Tip 24 e QuantStudio 3D Digital PCR Master Mix 1 5 mL 4485510 QuantStudio 3D Digital PCR 20K Chip Spares Kit e QuantStudio 3D Digital PCR Chip Case Lid 12 e QuantStudio 3D Digital PCR Sample Loading Blade 12 4485513 QuantStudio 3D Digital PCR Accessories Kit for GeneAmp PCR System 9700 e QuantStudio 3D Digital PCR Chip Carrier for Dual Flat Block GeneAmp PCR System 9700 2 e QuantStudio 3D Digital PCR Thermal Pad for Dual Flat Block GeneAmp PCR System 9700 2 4482592 QuantStudio 3D Digital PCR Chip Loader 4482710 QuantStudio 3D Digital PCR Master Mix Kit 1 5 mL 4483658 QuantStudio 3D Digital PCR Chip Loader 4485718 QuantStudio 3D Digital PCR Master Mix K
41. ata are reported in copies per uL along with the results of the quality assessment displayed using colored flags QuantStudio 3D Digital PCR System User Guide 63 Analyze the Prepared Chips Analyzing the data S asat ry About the results TM The QuantStudio 3D Digital PCR Instrument displays a summary of the imaging data after each successful run For each QuantStudio 3D Digital PCR 20K Chip imaged the instrument displays the following information screen Oo O 64 B0498N 166 7 cul 138 8 c uL Chip ID Displays the ID number of the Digital PCR 20K Chip If the instrument cannot read the chip ID the touchscreen displays the instrument name followed by the time and date of the run Time date stamp Displays the date and time of the run in lt yymmdd gt _ lt hhmmss gt format FAM VIC copy number estimate Displays the estimated concentration in copies per uL calculated from the fluorescence signal associated with the amplification of the nucleic acid sequence targeted by the FAM and or VIC dye labeled probes Beside the copy number estimate the QuantStudio 3D Instrument displays a flag icon color coded to indicate the status of the preliminary analysis of the image data for the associated probe Network status icon Represents the status of the QuantStudio 3D Instrument network connection e Wired Ethernet connection selected connected to network e amp Wired Ethernet
42. ate data destinations for the QuantStudio 3D AnalysisSuite Cloud Software for direct upload or for local Windows based or SAMBA file servers for later import You can create an unlimited number of destinations and select from them prior to each run receive run data 1 From the Main Menu of the touchscreen touch 2 In the Settings menu touch Data Destinations SETTINGS Instrument Settings Data Destinations Maintenance amp Service Run History QuantStudio 3D Digital PCR System User Guide 87 a res Appendix B Networking Connect the instrument to the network 3 In the Edit Destinations screen touch Add to add a data destination Note To edit an existing destination touch the name of the destination 4 EDIT DESTINATIONS Test Server Instrument SAMBA Fileshare Life Technologies 4 In the Choose Destination Type screen touch the desired network destination e Cloud Touch to specify a QuantStudio 3D AnalysisSuite Cloud Software data destination e LAN Touch to specify a network file server data destination Choose Destination Type 5 Touch each field enter the appropriate data for the destination then touch Save e Cloud data destination fields Target Name Enter a name to identify the cloud data destination Note The target name appears within the Destination Selection and Export screens User Name Enter the user name that the instrument sh
43. ated cover and thermal pads is uniform across all of the loaded chips QuantStudio 3D Digital PCR System User Guide 53 2 Ses 54 Perform the PCR Thermal cycle the Digital PCR 20K Chips b Lay the QuantStudio Thermal Pads over the Digital PCR 20K Chips IMPORTANT Make certain that each QuantStudio Thermal Pad is centered on the sample block and that it completely covers the Digital PCR 20K Chips IMPORTANT Do not allow the QuantStudio Thermal Pads to become dirty Fluids debris and other contaminants that adhere to the pad can transfer to the Digital PCR 20K Chips during thermal cycling and fluoresce during imaging e eS es ea c Slide the heated cover forward and pull the heated cover lever down to engage the cover with the QuantStudio 3D Digital PCR 20K Chips IMPORTANT Pull the heated cover completely forward to ensure a proper seal 4 From the Main menu of the GeneAmp PCR System 9700 press Run F1 08 00 AM 4 25 13 GeneAmp PCR System 9700 Version 3 12 Name tc001 User lt lt pe gt gt Run Create Edit Util User From the Stored Methods screen use the Circular Key Pad to select the method that you created previously then press Start Methods on Inst User Size Stored XL PCR lt lt pe gt gt LL 9 9 13 Touchdown PCR lt lt pe gt gt 6 9 9 13 AmpliTaq Gold lt lt pe gt gt 9 9 9 13 AmpliCycle Seq lt lt pe gt g
44. bp where m is the genome mass in grams and n is the genome size in base pairs The following example calculates the mass of the human genome using the Celera Genomics estimate of 3 0 10 bp haploid m 3 0 x 10 bp 1 096 x 10 g bp m 3 3 x 10 g or 3 3 pg The example is relevant to any gene that is present at the normal rate of two copies per diploid genome such as RNase P and provides a basis to perform a digital screening experiment to determine the optimal digital range QuantStudio 3D Digital PCR System User Guide 29 gt Prepare Samples and Load Reactions ms fei Lr Prepare the digital PCR reactions Determine the If the target copy number per genome is unknown say for a locus of unknown copies optimal dilution per genome or RNA of unknown expression level we recommend that you determine when the target is unknown the optimal dilution by loading and imaging a dilution series of the each sample at the expected digital range By assaying three to four data points above and below the expected digital range you ensure that one of the data points is within the optimal digital range Should real time data be available for the assay and sample being used this can guide the starting and end point of the dilution series Prepare the digital PCR reactions Required The following materials are required to prepare digital PCR reactions for use on the materials QuantStudio 3D Digital PCR System TaqM
45. cant force to decouple If necessary insert a flathead screwdriver into the slot at the base of the panel to pry it from the instrument chassis insert it into the USB port within the instrument When the device is seated within the USB port firmly push on the wireless dongle to ensure that it is plugged in completely The internal USB port is accessed through a small opening in the instrument chassis Reattach the rear panel to the instrument chassis then plug in and power on the QuantStudio 3D Instrument 2 From the Main Menu of the touchscreen touch 2 then touch Instrument Settings QuantStudio 3D Digital PCR System User Guide 3 Choose the appropriate option Appendix B Networking Connect the instrument to the network If connecting your instrument to a Action Wired network that supports DHCP Network setup is complete Note The QuantStudio 3D Instrument is pre configured for DHCP operation and automatically joins the network when connected e Wireless network e Wired network with specific requirements such as static IP operation Go to step 4 4 In the Instrument Settings screen touch Network Setup then touch Edit in the network setup screen 4 Wired Connected DHCP oat 10 10 10 10 255 255 252 0 10 10 10 10 5 Wireless connection only If you are connecting your instrument to a wireless network a In the N
46. ctions on securing your methods want to return to the Main menu without press Cancel F5 adding the new user name Connect the QuantStudio 3D Instrument to a network TM If you choose not to connect the QuantStudio 3D Instrument using the setup wizard during installation you can connect it to a network at any time afterwards to streamline data transfer to the QuantStudio 3D AnalysisSuite Software If you choose to connect your QuantStudio 3D Instrument see Appendix B Networking for the complete installation procedure including guidelines for integrating the instrument into your laboratory network IMPORTANT This document does not provide adequate detail to integrate the QuantStudio 3D Instrument into all possible network architectures Because your laboratory network can contain advanced features such as firewalls Life Technologies recommends that you consult a network administrator before connecting your instrument Connect the QuantStudio 3D AnalysisSuite Software You can connect to the QuantStudio 3D AnalysisSuite Software before or after you install the QuantStudio 3D Digital PCR Instrument The software supports two methods of deployment cloud and server so the method that you follow will depend upon the deployment you purchased Regardless of the deployment you can use the AnalysisSuite Software web application to analyze data generated by your QuantStudio 3D Instrument aft
47. ctrical requirements Environmental requirements Ensure that the installation room is maintained under correct environmental conditions Note See the GeneAmp PCR System 9700 Base Module User s Manual Pub no 4303481 for the GeneAmp PCR System 9700 environmental requirements Condition Acceptable Range Installation site Indoor use only Altitude Located between sea level and 2000 m 6500 ft above sea level Humidity 20 80 noncondensing QuantStudio 3D Digital PCR System User Guide 95 Specifications and Layout Network requirements Condition Acceptable Range Temperature 15 C to 30 C 60 F to 85 F Thermal output During operation the net thermal output based on the actual current draw of the QuantStudio 3D Instrument is expected to be approximately 12 W 41 BTU hr Vibration Ensure the instrument is not adjacent to strong vibration sources such as a centrifuge pump or compressor Excessive vibration will affect instrument performance Pollution The instrument has a Pollution Degree rating of Il The instrument may only be installed in an environment that has nonconductive pollutants such as dust particles or wood chips Typical environments with a Pollution Degree II rating are laboratories and sales and commercial areas The noise output of the instrument is 0 dB at idle Other conditions Ensure the room is away from any vents that cou
48. cts Instrument safety General AN CAUTION Do not remove instrument protective covers If you remove the protective instrument panels or disable interlock devices you may be exposed to serious hazards including but not limited to severe electrical shock laser exposure crushing or chemical exposure Electrical AN WARNING Fuse Installation Before installing the instrument verify that the fuses are properly installed and the fuse voltage matches the supply voltage Replace fuses only with the type and rating specified for the unit Improper fuses can damage the instrument wiring system and cause a fire WARNING Ensure appropriate electrical supply For safe operation of the instrument Plug the system into a properly grounded receptacle with adequate current capacity Ensure the electrical supply is of suitable voltage Never operate the instrument with the ground disconnected Grounding continuity is required for safe operation of the instrument WARNING Power Supply Line Cords Use properly configured and approved line cords for the power supply in your facility WARNING Disconnecting Power To fully disconnect power either detach or unplug the power cord positioning the instrument such that the power cord is accessible QuantStudio 3D Digital PCR System User Guide 101 Appendix E Safety Safety and electromagnetic compatibility EMC standards Cleaning and decontamination
49. cycling of Digital PCR 20K Chips must be performed on a Dual Flat Block GeneAmp PCR System 9700 as described in this guide No other thermal cyclers can be substituted at the time of the release of this manual 1 Open the GeneAmp PCR System 9700 heated cover then wipe the surface of both sample blocks using a lint free wipe to ensure that they are clean and dry 2 Confirm the Tilt Base is installed to the GeneAmp PCR System 9700 and that Chip Carriers are installed to both sample blocks IMPORTANT You must install Chip Carriers to both sample blocks when thermal cycling Digital PCR 20K Chips even if you are using only one of the blocks 3 Load the QuantStudio 3D Digital PCR 20K Chips into the GeneAmp PCR System 9700 a Place the sealed Digital PCR 20K Chips onto the sample block so that the fill ports on the chips are positioned toward the front of the thermal cycler The fill port must be elevated during thermal cycling to prevent leaks from improperly sealed chips IMPORTANT If you are thermal cycling less than 24 Digital PCR 20K Chips load according to the following guidelines Load each Digital PCR 20K Chip so that the fill port is oriented toward the front of the GeneAmp PCR System 9700 O EEA Front of instrument Load the right sample block first placing at least 1 chip on the right sample block Balance the load between the left and right blocks so that the pressure applied by the he
50. dio 3D Digital PCR Instrument fail perform the following procedure to replace them Materials d required Fuses 1 6A Time Lag T 250V AC 5 x 20 mm 2 Safety glasses Powder free gloves Screwdriver flathead QuantStudio 3D Digital PCR System User Guide 75 Maintenance Update the instrument firmware Replace the fuses Ad 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 QuantStudio 3D Instrument 1 Power off then unplug the QuantStudio 3D Digital PCR Instrument 2 Using a flat head screwdriver pry open the fuse door and remove the fuse holder 3 Remove each fuse from its fuse holder and inspect it for damage Carbon typically coats the inside of failed fuses 4 Replace each failed fuse with a 1 6A Time Lag T 250VAC 5 x 20 mm Fuse Note The voltage and amperage ratings are on the fuse holder 5 Install the fuse holder 6 Plug in then power on the QuantStudio 3D Instrument The installation is successful if the instrument powers on Note Fuse failure can result from fluctuations in the supplied power to the QuantStudio 3D Instrument To prevent further failures consider installing an electrical protective device such as a UPS or surge protector Update the instrument firmware TM You can download QuantStudio 3D Digital PCR Instrument firmware updates
51. e BS Seen eG a SiG EMS ee 101 Instrument Safety rorta reaa reai Ae ea eked ext eeee sa aaa a a aa a Pies eas 101 General aa sas ds ao a ds ead belie ee Geet oo 101 Electrical mi iaae eg cot a eta oie eee tt ai a a he nh eee ear 101 Cleaning and decontamination 200 c cece eee eee 102 Safety and electromagnetic compatibility EMC standards 0005 102 Safety iret ater geceen tye ee Basie sextet ee Eek E a A E e EE E S ee n ated 102 EME eeoa E E E EE ete AAE EA eee oe 102 Environmental design seasga areia puria e eee eee eee eee eee eee 103 Chemicalisafety unisi e a Athe sue aes ee Ad eS ee ae Ae aes 103 Biological hazard safety 0 2 c cece eee eens 104 Documentation and support cece eee eee e cent eee eeeeeeeeees 105 Obtaining SDSS ia 5h e ae ead ge eta p aa amp eels tia g beaded Med 105 Obtaining Certificates of Analysis 0 00 00 eee eee 105 Obtaining SUppPOrt 3 12 22 c2nqsed eee hone eee nes wheat keel a lt ieee eet 105 Limited product warranty 200 cece eee eee 105 QuantStudio 3D Digital PCR System User Guide About This Guide AN CAUTION ABBREVIATED SAFETY ALERTS Hazard symbols and hazard types specified in procedures may be abbreviated in this document For the complete safety information see the Safety appendix in this document IMPORTANT Before using this product read and understand the information in the Safety appendi
52. e appropriate action IMPORTANT The QuantStudio 3D Instrument is pre configured for DHCP network operation If connecting your instrument to a network that requires static IP assignment or advanced network configuration touch Skip Network Connection and connect your instrument to the network following the installation e Touch Connect to Network to connect the QuantStudio 3D Instrument to a wired or wireless network that supports DHCP Go to substep 4 e Touch Skip Network Connection if you do not want to connect to a network if you want to connect the instrument at a later time or if your network requires static IP assignment or advanced network settings Go to step 8 Network Connection Connect to Network Skip Network Connection QuantStudio 3D Digital PCR System User Guide Getting Started Prepare the QuantStudio 3D Digital PCR System for use d In the Choose Connection Type screen select the appropriate connection Connection Action Wired Touch LAN then wait for the instrument to connect to the network Wireless 1 Touch Wireless 2 In the Available Wireless Network screen touch the desired wireless hotspot Note The signal strength of a wireless hotspot is indicated by the number of bars present in the wireless icon The presence of a lock icon indicates that the hotspot is secure WPA WEP or WPA2 Q Lifetech Available Wireless Network Life
53. e de conducteur de protection ground mise a la terre principale QuantStudio 3D Digital PCR System User Guide Appendix E Safety Symbols on this instrument Symbol English Francais xt Do not dispose of this product in unsorted municipal waste CAUTION To minimize negative environmental impact from disposal of electronic waste do not dispose of electronic waste in unsorted municipal waste Follow local municipal waste ordinances for proper disposal provision and contact customer service for information about responsible disposal options Ne pas liminer ce produit avec les d chets usuels non soumis au tri s lectif CAUTION Pour minimiser les cons quences n gatives sur l environnement a la suite de l limination de d chets lectroniques ne pas liminer ce d chet lectronique avec les d chets usuels non soumis au tri s lectif Se conformer aux ordonnances locales sur les d chets municipaux pour les dispositions d limination et communiquer avec le service a la client le pour des renseignements sur les options d limination responsable Conformity symbols mark Conformity Description Indicates conformity with safety requirements for Canada and U S A electromagnetic compatibility Indicates conformity with European Union requirements for safety and compatibility Indicates conformity with Australian standards for
54. e spaces or special characters lt gt 1 Note By default the Instrument Name is set as the serial number of the instrument e Touch the Time Zone field then select the correct continent and region from the list e Touch the Date field enter the current date then touch Enter e Touch the Time field enter the appropriate time units then touch Enter 20 QuantStudio 3D Digital PCR System User Guide Getting Started Prepare the QuantStudio 3D Digital PCR System for use SAD h In the Configuration screen touch OK to accept the instrument configuration The QuantStudio 3D Instrument is ready to use when the touchscreen displays the Start Run screen Start Run Start Run 8 Unpack and place the GeneAmp PCR System 9700 Base Module a Open the package containing the GeneAmp PCR System 9700 Base Module b Remove the packing crate from the GeneAmp PCR System 9700 Package and verify that it contains all of the parts listed on the shipping manifest If you have not received one or more of the parts contact Life Technologies for replacement c Remove the GeneAmp PCR System 9700 Base Module from the package and set it on a clean level surface then remove the protective cover J 15 2 cm gt 6 in _ p 1 20 cm LL 8 in 2 in QuantStudio 3D Instrument GeneAmp PCR Sys
55. eee eee eee 12 Prepare the QuantStudio 3D Digital PCR System for use 0 eee eee eee es 13 About the installation 00 0 cece eee eee 13 Materials required o 6cc cecc coer egawr nde iie ep a ai Pane eae a G 13 Before you begin 0 c aa eee eee 13 Plan the laboratory layout 0 ccc ccc cece cece eee eee a n E 13 Choose additional protective devices 000 eee eee eee eee eee 15 Install the QuantStudio 3D Digital PCR System 0cceceeeeeeeeeees 15 Configure the GeneAmp PCR System 9700 settings 2 eee eeee 25 Connect the QuantStudio 3D Instrument to a network cceeeeeeeeeeeeeeees 27 Connect the QuantStudio 3D AnalysisSuite Software eee e eee eee 27 m CHAPTER2 Prepare Samples and Load Reactions 28 Prepare the DNA samples 0 020 c eee eee ee eet eee 28 Quality of DNA s viewer ioddtoea bc ee teed a a E E a a 28 Quantity Of DNA eeiam ce ied San be aa e acd ae e A hontai aE a eet EE A E ae androud ants 28 Sample Gitlin 2 ccc dcncccwc ewe seachgusee cies coat ee a wae aa eee 29 Determine the optimal dilution when the target is known 22 2 00 29 QuantStudio 3D Digital PCR System User Guide 3 Contents Determine the optimal dilution when the target is unknown 30 Prepare the digital PCR reactions 0 cece eee eee eens 30 Required materials diein ai aaa
56. eee eens 30 Guidelines for PCR sample preparation 2 0 0c e eee eee eee 30 Prepare the reaction mix and samples 2 0 cee eee eee ee eee eee 31 Load the Digital PCR 20K Chips 0 e eee e eee eee eee eens 32 Guidelines for loading and sealing Digital PCR 20K Chips 32 Load the chips using the Chip Loader 002 c cece eee eee ee eee 33 Load chips manually 0 0 0 0 cece ee eee eens 41 CHAPTER3 Perform the PCR cee eeeee cent teen eeeeeeee 49 Choosing a thermal cycler 0 cece eee eee eee eee 49 About the Dual Flat Block GeneAmp PCR System 9700 00 02 ee eee 49 Prepare the GeneAmp PCR System 9700 2 0 cee eee eee 50 Thermal cycle the Digital PCR 20K Chips 0 0 22 cece eee eee ee eee 52 Materials required 00 c cece eee eens 52 Guidelines for handling Chip Cases 000 cece eee eee 52 Perform the PCR 2 0 30 f seh acy Sacha oie hy pare E E we aisle nid geen Sie 53 Unload the thermal cycler 0 ccc cece cece eee te dne EE teen eeeees 55 CHAPTER 4 Analyze the Prepared Chips 0eceeceeeeeees 57 Using the QuantStudio 3D Digital PCR Instrument 0ceeeeeeeeeeeaees 57 About imaging and primary analysis 0 e eee eee eee ees 57 Analysis workflow 2 000 cece ee eee ee eee eee eee eee eens 58 About the instrument interface
57. electromagnetic QuantStudio 3D Digital PCR System User Guide 99 Appendix E Safety Safety alerts on this instrument Safety alerts on this instrument Location of safety alerts on the instrument 100 Additional text may be used with one of the symbols described above when more specific information is needed to avoid exposure to a hazard See the following table for safety alerts found on the instrument English French translation Location on Instrument CAUTION Hazardous chemicals Read the Safety Data Sheets SDSs before handling ATTENTION Produits chimiques dangereux Lire les fiches signal tiques FS avant de manipuler les produits Rear instrument panel CAUTION Hazardous waste Refer to SDS s and local regulations for handling and disposal ATTENTION D chets dangereux Lire les fiches signal tiques FS et la r glementation locale associ es a la manipulation et a l limination des d chets QuantStudio 3D Digital PCR System User Guide Appendix E Safety Safety information for instruments not manufactured by Life Technologies Cor poration Safety information for instruments not manufactured by Life Technologies Corporation Some of the accessories provided as part of the instrument system are not designed or built by Life Technologies Corporation Consult the manufacturer s documentation for the information needed for the safe use of these produ
58. ells The 20 000 physical cycling and imaging holes within the Digital PCR 20K Chip QuantStudio 3D Digital PCR Chip Case that suspend the individual PCR The thermal conductive base that reactions secures and protects the Digital PCR 20K Chip during all phases of use QuantStudio 3D Digital PCR System User Guide 9 Getting Started m About the QuantStudio 3D Digital PCR System 0 0 0 0000s 10 m Operating the instrument 6 0 c cee een ees 12 m Prepare the QuantStudio 3D Digital PCR System for use 00 13 E Connect the QuantStudio 3D Instrument to a network 000 27 E Connect the QuantStudio 3D AnalysisSuite Software 0 27 About the QuantStudio 3D Digital PCR System TM The QuantStudio 3D Digital PCR System uses fluorescent labeled probe based quantitative polymerase chain reaction PCR reagents to provide quantitative and qualitative research detection of target nucleic acid sequences targets using post PCR endpoint analysis The QuantStudio 3D Instrument performs imaging and primary analysis of QuantStudio 3D Digital PCR 20K Chips that have been loaded with TaqMan assays and thermal cycled using a Dual Flat Block GeneAmp PCR System 9700 Secondary analysis and post processing is performed by the QuantStudio 3D AnalysisSuite Software to yield relative or absolute quantification results from the raw imaging data The fo
59. ent a Open the QuantStudio 3D Digital PCR Instrument Package b Confirm that you have received all parts listed on the shipping manifest If you have not received one or more parts contact Life Technologies for a replacement c Remove the QuantStudio 3D Instrument from the package and set it on a clean level surface then remove the protective cover d Inspect the QuantStudio 3D Instrument for damage caused during transportation If the instrument is damaged note the location and appearance of the damage then contact Life Technologies support or your service representative for assistance 16 QuantStudio 3D Digital PCR System User Guide Getting Started Prepare the QuantStudio 3D Digital PCR System for use 6 If you intend to connect your instrument to a network prepare the physical network connection IMPORTANT Connecting the instrument to a network is optional and can be done at a later time if you desire Connection Action Wired Connect one end of a standard category 6 Ethernet cable to the RJ 45 port on the back panel of the instrument and the other end to an open network port Wireless 1 If required for network setup record the MAC address of the SparkLAN WUBR 170GN 802 11n Single Band Wireless Dongle which is located on the underside of the device 2 Unplug the QuantStudio 3D Instrument 3 Turn the QuantStudio 3D Instrument over then use your fingers to carefully pr
60. ent must be accessible at all times In case of emergency you must be able to immediately disconnect the main power supply to all the equipment Allow adequate space between the wall and the equipment so that the power cords can be disconnected in case of emergency IMPORTANT Use only the approved power supply provided by Life Technologies to power the QuantStudio 3D Digital PCR Chip Loader e Electric receptacle is required e Mains AC line voltage tolerances must be up to 10 percent of nominal voltage Note Place the instrument power receptacle 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 15A circuit is required i Rated Rated Rated Rated Device voltage frequency current power QuantStudio 3D Digital PCR 100 50 60 Hz 150 mA 10 VA Instrument 240 VAC QuantStudio 3D Digital PCR 12 VDC 2 5 A max Chip Loader Dual Flat Block GeneAmp 2 PCR System 9700 11 If the supplied power fluctuates beyond the rated voltage a power line regulator may be required High or low voltages can adversely affect the electronic components of the instrument 21 See the GeneAmp PCR System 9700 Base Module User s Manual Pub no 4303481 for the GeneAmp PCR System 9700 ele
61. ents IEC 61010 1 UL 61010 1 IEC CSA UL 61010 1 IEC 61010 2 010 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 010 Particular requirements for laboratory equipment for the heating of materials 1 Applies only to the QuantStudio 3D Digital PCR Chip Loader Reference Description Directive European Union EMC Directive 2004 108 EC EN 61326 1 Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Part 1 General Requirements FCC Part 18 47 CFR U S Standard Industrial Scientific and Medical Equipment QuantStudio 3D Digital PCR System User Guide Environmental design Chemical safety Appendix E Safety Chemical safety Reference Description AS NZS 2064 Limits and Methods of Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radiofrequency Equipment ICES 001 Issue 3 Industrial Scientific and Medical ISM Radio Frequency Generators Reference Description Directive 2002 96 EC European Union WEEE Directive Waste electrical and electronic equipment WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precau
62. er switch for the instrument functions such as data transfer and instrument operation Chip tray Conveys the QuantStudio 3D Digital PCR 20K Chip to and from the imaging stage in the interior of the instrument USB port Provides USB communication with the QuantStudio 3D Instrument Can be used to transfer data to and from the instrument instrument where the states are on oroff 0 Power port A 100 240 VAC port that provides power to the QuantStudio 3D Instrument Ethernet port An RJ45 port that provides Ethernet gigabit communication with the QuantStudio 3D Instrument Access panel Provides access to the interior of the QuantStudio 3D Instrument Fuse cover Dual 1 6A Time Lag T 250 VAC 5 x 20 mm electrical fuses that protect the instrument from excessive electrical current Instrument software The QuantStudio 3D Digital PCR Instrument includes a software suite used to analyze and manage digital PCR data generated by imaging QuantStudio 3D Digital PCR 20K Chips The QuantStudio 3D AnalysisSuite Software features a server and cloud deployment that can satisfy a broad range of laboratory requirements Note For more information on the AnalysisSuite Software refer to the AnalysisSuite Software Help which is available from any point in the software by clicking the button The QuantStudio 3D AnalysisSuite Software is accessed as a web based application that is optim
63. er you connect it to your network For instructions on using the AnalysisSuite Software see the QuantStudio 3D AnalysisSuite Software Help QuantStudio 3D Digital PCR System User Guide 27 a y A 4 K N M ze 3 Prepare Samples and Load Reactions E Prepare the DNA samples 0060s 28 E Prepare the digital PCR reactions 6 eens 30 E Load the Digital PCR 20K Chips 20 0 ccc eee eee 32 Prepare the DNA samples Quality of DNA Quantity of DNA 28 Life Technologies recommends the following best practices for the preparation of DNA template for use in digital PCR experiments Because digital PCR experiment strategy and methodology can vary significantly sample preparation and template quality must be assessed on an individual basis Make sure that the DNA you use for experiments Is extracted from the raw material that you are testing with an optimized protocol salting out procedures and crude lysates are not recommended Does not contain PCR inhibitors Has A260 230 and A260 280 ratios between 1 7 and 1 9 Note The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA A ratio of 1 8 is generally accepted as pure for DNA a ratio of 2 0 is generally accepted as pure for RNA If the ratio is appreciably lower in either case it may indicate the presence of protein phenol or other contaminants that absorb strongly at or near 280 nm The ratio
64. et them on a clean dry surface IMPORTANT Do not discard the QuantStudio Thermal Pads They can be reused multiple times Remove the Chip Carriers from the GeneAmp PCR System 9700 sample block and place them on a clean dry surface Remove the Digital PCR 20K Chips from the Chip Carriers and allow them to equilibrate to room temperature Note To remove a chip from a Chip Carrier push the Digital PCR 20K Chip from beneath and grasp the edges of the Chip Case then remove it IMPORTANT Handle the Chip Cases gently being careful to not apply pressure to the Digital PCR 20K Chips within them QuantStudio 3D Digital PCR System User Guide 55 56 Perform the PCR Thermal cycle the Digital PCR 20K Chips 4 Visually inspect each Digital PCR 20K Chip for signs of leaks or potential problems Using a lint free laboratory wipe remove any condensation or Immersion Fluid from the surface of the chip by wiping in one direction If necessary use a lint free laboratory wipe sprayed with isopropanol to remove any dried residue IMPORTANT Thoroughly clean each Chip Case before proceeding The Digital PCR 20K Chip is now ready to be imaged by the QuantStudio 3D Instrument Run the prepared Digital PCR 20K Chips within 1 hour after removing them from the thermal cycler IMPORTANT Store the prepared Digital PCR 20K Chips in a clean dry dark location until you are ready to image them Prolonged exposure to light c
65. etwork Setup screen touch Change Connection Type b In the Choose Connection Type screen touch Wireless c Inthe Available Wireless Network screen touch the desired wireless hotspot Note The signal strength of a wireless hotspot is indicated by the number of bars present in the wireless icon The presence of a lock icon indicates that the hotspot is secure WPA WEP or WPA2 4 Available Wireless Network Lifetech Lifetech Guest Join Other Network QuantStudio 3D Digital PCR System User Guide 83 84 Appendix B Networking Connect the instrument to the network d If your selected wireless hotspot is secure enter the required authentication information user name password then touch OK Enter Username amp Password This network requires username and password If connecting your instrument to a network that supports DHCP network setup is complete Otherwise go to the next step to configure the TCP IP settings for the instrument TM 6 Set the internet protocol TCP IP properties for the QuantStudio 3D Instrument a In the Network Setup screen touch Edit Settings b In the Network Setup settings touch the IP Configuration field touch the setting appropriate for your network Static IP or DHCP then touch OK e Static IP Select if you have a static IP address to assign to the instrument e DHCP Select to allow your network DHCP server to automatically assign a
66. g Blade to the Digital PCR 20K Chip more than once e Allow the Digital PCR 20K Chip to sit on the heated block for 20 seconds while the PCR reaction stabilizes within the chip f Add 10 15 drops of Immersion Fluid directly to the chip so that the fluid covers the entire surface IMPORTANT Do not allow the syringe tip to contact the Digital PCR 20K Chip Note A small amount of fluid running off the surface of the chip is acceptable 7 Seal the Digital PCR 20K Chip a Remove the red protective film from the QuantStudio 3D Digital PCR Chip Case Lid then carefully place it onto the prepared Digital PCR 20K Chip so that the window in the lid aligns with the chip IMPORTANT Before applying the Chip Case Lid make certain that it is correctly oriented to the Digital PCR 20K Chip assembly QuantStudio 3D Digital PCR System User Guide 45 46 Prepare Samples and Load Reactions Load the Digital PCR 20K Chips b Pinch together all four sides and corners of the Chip Case for at least 10 seconds to seal it While pressing apply some lateral pressure to ensure a tight seal Note You can remove the Digital PCR 20K Chip from the loading surface when you apply pressure to seal it x Je 9E000E0X Ie 8 Fill the Chip Case with Immersion Fluid a Hold the Chip Case by its edges and at a 45 angle so that air can escape from the loading port as you fill it then carefully dispense Immersion Flu
67. g in the run Note While the run is in progress you can stop the run at any time by touching Cancel Note The touchscreen reports the QuantStudio 3D Instrument status as USB in use while the instrument writes the results to the USB drive When the touchscreen displays the Analyzing Chip screen open the chip tray and remove the Digital PCR 20K Chip Store or discard it as desired Analyzing Chip Analyzing Do not remove USB Data You may insert another chip You can read another Digital PCR 20K Chip without waiting for the analysis to complete To begin the next run load the chip and touch Start Run as described in step 3 Note Running chips in rapid succession will not compromise the ongoing analysis however the QuantStudio 3D Instrument performance may slow slightly as more chips are read The QuantStudio 3D Instrument displays the results of the chip analysis after it has processed the imaging data and transferred the results to the selected destination QuantStudio 3D Digital PCR System User Guide Analyzing the data About the analysis Analyze the Prepared Chips irae i Analyzing the data as 6 After reviewing the results of the run touch Done to close the results screen B0498N 166 7 C uL 138 8 C uL If the analysis of the imaging data produced a red for either probe FAM or VIC dye visually inspect the chip for problems and read the chip again Note If the run fa
68. g temp Config Instrument configuration Diag TmCalc Config More Exit 3 From the Instrument Configuration screen set the instrument time a b Use the Circular Key Pad to select the Time field Press the 24 Hr F2 or PM AM F3 soft keys until the format you want for the current time displays in the Time field Use the numeric keys to type in the hours followed by minutes Press Accept F1 when your entries are complete Instrument Configuration Time 11 30 AM Date 01 25 00 M D Y Run Time Printer Off Run Time Beep oft Accept 24Hr PM More Cancel Note CE clears an entry 4 From the Configuration screen set the instrument date a b Use the circular key to select the Date field Press the D M Y or Y M D soft keys until the format you want for the current date displays in the Date field Use the numeric keys to enter the values into the Day Month and Year fields QuantStudio 3D Digital PCR System User Guide 25 Re 26 Getting Started Prepare the QuantStudio 3D Digital PCR System for use Add new users The GeneAmp PCR System 9700 stores methods by user s names You can add up to 19 different user names to the instrument Once you ve added your name to a list of users you can store methods under that name and run them at any time from the Stored Methods screen 1 From the Main menu press User F5 The
69. gently tap it to remove bubble before loading Chip view Chip view e Excess PCR reaction was present on the Digital PCR 20K Chip after loading it with the Sample Loading Blade e The Sample Loading Blade was drawn across the chip too quickly or at an angle shallower than 70 80 If possible use the AnalysisSuite Software to filter the low quality data points or discard the chip and run the sample again To prevent leaving excess PCR reaction on the Digital PCR 20K Chips e Confirm that the heated block used to load Digital PCR 20K Chips is set to 40 1 C e Make sure to wait 20 seconds after loading a Digital PCR 20K Chip before pre wetting it with Immersion Fluid e Make sure to install theTilt Base to the GeneAmp PCR System 9700 You must thermal cycle the Digital PCR 20K Chips at a 11 angle e Make sure to draw the Sample Loading Blade across the chip slowly gt 10 seconds and ata 70 80 angle 70 QuantStudio 3D Digital PCR System User Guide Chapter 5 Troubleshooting 5 Troubleshooting chip images using the Chip View z2 Observation Possible Cause Action Chip view Chip view The Digital PCR 20K Chip leaked during thermal cycling or imaging A large bubble was present in the chip insufficient Immersion Fluid Immersion Fluid was not applied to the chip immediately after loading evaporation of the PCR reaction Exce
70. gital PCR Chip Loader then wait until the Chip Loader status light illuminates solid green lt 20 minutes indicating that the chip nest has reached operating temperature b Open the QuantStudio 3D Digital PCR 20K Chip package then gently grasp the chip by its sides and load it face up into the chip nest Lock the Digital PCR 20K Chip into place by pressing down the chip nest lever prior to placing the chip into the chip nest as shown IMPORTANT Confirm that the Digital PCR 20K Chip is correctly aligned within the Chip Loader IMPORTANT If you do not intend to load an opened Digital PCR 20K Chip immediately cover the chip using the aluminum plate found within the packaging to prevent contamination Note If desired use a pen to label the back of the Digital PCR 20K Chip Marking the back of the chips will not affect the imaging data QuantStudio 3D Digital PCR System User Guide Prepare Samples and Load Reactions 5 Load the Digital PCR 20K Chips c Press the sample loading blade lever then install the QuantStudio 3D Digital PCR Sample Loading Blade to the Chip Loader as shown Press down on the body of the Sample Loading Blade to confirm that the blade is properly seated d Remove the red protective film from a QuantStudio 3D Digital PCR Chip Case Lid press the Lid Nest button carefully place the Chip Case Lid into the Lid Nest as shown then release the button to clamp the chip in place IM
71. h for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support QuantStudio 3D Digital PCR System User Guide 105 Headquarters o 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit lifetechnologies com support or email techsupportfdlifetech com technologies lifetechnologies com 16 December 2013
72. hips are loaded To maximize battery life make sure to power off the UV Stylus after the last chip has been sealed c Load the Digital PCR 20K Chip assembly into the UV Stylus Stand and allow the Chip Sealant to cure for 0 5 3 minutes A longer exposure no longer than 3 minutes ensures a complete seal IMPORTANT Make sure that the fill port is located directly beneath the beam of ultraviolet light Note If necessary you can illuminate the fill port for up to 3 minutes to ensure complete curing of the Chip Sealant QuantStudio 3D Digital PCR System User Guide 47 gt Prepare Samples and Load Reactions C Load the Digital PCR 20K Chips 10 Visually inspect the sealed Digital PCR 20K Chip for potential problems e Leaks Confirm that no Immersion Fluid is leaking from the fill port or around the seal between the Chip Case and lid e Bubbles Confirm that the Chip Case is free of excessive bubbles One small air bubble is acceptable e Correct lid orientation Confirm that the Chip Case Lid and the Digital PCR 20K Chip are correctly aligned If the loaded and sealed Digital PCR 20K Chip fails any of the criteria above discard it and prepare a duplicate chip Once sealed the Chip Case cannot be opened and resealed IMPORTANT Handle the Chip Cases gently being careful to not apply pressure to the Digital PCR 20K Chips within them Thermal cycle the prepared Digital PCR 20K Chips within 2 hours after
73. id into the port until the Chip Case contains an air bubble no larger than the fill port lt 2 3 mm in diameter IMPORTANT Expel the Immersion Fluid slowly If injected too quickly the fluid can flush the reactions suspended within the chip Note Minimize air bubble creation when you dispense the fluid however one small air bubble lt 2 3 mm in diameter is acceptable 9E000E0X IG b Using a lint free laboratory wipe remove any excess Immersion Fluid from TM the Chip Case to ensure optimal imaging on the QuantStudio 3D Instrument QuantStudio 3D Digital PCR System User Guide Prepare Samples and Load Reactions Load the Digital PCR 20K Chips 9 Seal the Chip Case using Chip Sealant a Insert the syringe tip into the fill port of the sealed Chip Case then carefully fill the port with Chip Sealant ensuring that the fluid touches the walls of the hole When full finish the seal by lifting the syringe tip and add a small amount of sealant over the top of the port IMPORTANT Apply the Chip Sealant to the fill port only Do not apply the sealant to the Chip Case Lid window IMPORTANT When not in use store the Chip Sealant inside its original protective package and with the syringe tip attached to prevent the sealant in the tip from curing 9 000 0X b Power on the UV Stylus in the UV Stylus Stand by twisting the cap near the power button Note The UV Stylus can remain on until all c
74. ies Corporation and or its affiliate s or their respective owners Microsoft and Windows are registered trademarks of Microsoft Corporation Macintosh is a registered trademark of Apple Inc TaqMan is a registered trademark of Roche Molecular Systems Inc used under permission and license 2013 Life Technologies Corporation All rights reserved Contents About UNIS BUNGE cis atte wstenene ensen a a a a 7 Revision RiStory secs d cede renni ranne aaa ine kd bad bade a E eld eed Wee ba 7 PUMPOSC oii oeine ct weosen ene Hamme E a es amid ced E SGN SR E E EE ine 7 PREREQUISITSS setoicirnieserise Bette pee nak wkd Ba eeed Damen OE Raa E 7 Instrument hardware 0 200 c cece eee ete 8 InstruMeNt SOfWAMNE 0 06 Gl eer keke ea eee oben bee hee aE eS 8 Computer requirements 0000 eee eee eee eee 8 Instrument consumables 0 0 0 e eee eens 9 About the Digital PCR 20K Chip 00 cece eee eee ees 9 mM CHAPTER 1 Getting Started ccc ccc cece eee e eee eeee canes 10 About the QuantStudio 3D Digital PCR System 00cceeeeeeeeeeeeeeeues 10 Data COlleCtiony iss srocn ashes erg tele Cee BER LE Re Se de SE eee eae 11 Instrument filters and supported dyes 0000 c eee eee eee eee eee eee 11 Operating the instrument 0 cece ee eee eee 12 Compatible reagent kits and consumables 00 0c cece eee eee eee 12 Operational workflow 2 0 ccc eee
75. ils because the instrument is unable to read the Digital PCR 20K Chip touch Done then touch Start Run again to repeat the reading If the run fails repeatedly discard the chip and prepare a replacement After a Digital PCR 20K Chip has been read e You run the imaged Digital PCR 20K Chip again for up to 1 hour after thermal cycling If you read multiple Digital PCR 20K Chips in rapid succession touch the scroll buttons E to review the results of the previously imaged chips The QuantStudio 3D Instrument retains a copy of the analyzed data for the imaged Digital PCR 20K Chip that you can access from the Run History screen Note The results will remain in the QuantStudio 3D Instrument cache for approximately 300 readings After 300 chips the instrument removes the oldest data file in the cache to store each new reading TM After the QuantStudio 3D Digital PCR Instrument images a Digital PCR 20K Chip the instrument performs an initial evaluation of the raw data to determine the viability of the collected data During the analysis the instrument identifies the wells within the captured images and performs a quality assessment of the composite results to determine whether the instrument has collected usable data If the imaging data pass the initial quality assessment the QuantStudio 3D Instrument calculates the estimated concentration of the nucleic acid sequence targeted by the FAM and VIC dye labeled probes The resulting d
76. ilt Base for Dual Flat Block GeneAmp PCR System 9700 and install it beneath the thermal cycler 22 QuantStudio 3D Digital PCR System User Guide Getting Started Prepare the QuantStudio 3D Digital PCR System for use SADU b Open the containing the QuantStudio 3D Digital PCR Chip Carrier for Dual Flat Block GeneAmp PCR System 9700 and install them Note Each Chip Carrier includes a set of four alignment pegs that fit into the holes of a flat sample block The Chip Carrier can fit onto either side of the sample block right or left and in any orientation c Connect the GeneAmp PCR System 9700 power cord power on the thermal cycler then wait for it to start about 30 sec The cooling fan powers up and the start up screens appear The GeneAmp PCR System 9700 is ready to use when the Main Menu displays 08 00 AM 4 25 13 GeneAmp PCR System 9700 Version 3 12 Name tc001 User lt lt pe gt gt Run Create Edit Util User IMPORTANT If the GeneAmp PCR System 9700 does not start check the position of the Dual Flat Block Module The instrument cannot be powered on if the module is seated incorrectly IMPORTANT If the GeneAmp PCR System 9700 does not start even after adjusting the Dual Flat Block Module or if the screen contains any permanent patterns of lines or bars contact Life Technologies support or your service represe
77. intain strict physical isolation between the area designated for handling amplified DNA and the other area thereby avoiding transfer of amplified DNA out of the designated work area QuantStudio 3D Digital PCR System User Guide 13 Bick 3 E fa 4 a Y C Getting Started Prepare the QuantStudio 3D Digital PCR System for use Room Activities Equipment PCR Setup Work e Sample preparation and DNA General laboratory supplies such as gloves and Area pre PCR extraction wipes room e Extracted DNA sample storage Microcentrifuge e PCR reaction preparation and Vortexer DNA sample additions Pipettes P10 to P1000 with tips e QuantStudio 3D Digital PCR 20K QuantStudio 3D Digital PCR 20K Chip Chip loading and sealing m r QuantStudio 3D Digital PCR Chip Case Lid QuantStudio 3D Digital PCR Sample Loading Blade QuantStudio 12K Flex OpenArray Immersion Fluid UV Activated Chip Sealant Syringes and Tips UV Curing Stylus for Chip Sealant and Stand Amplified DNA e PCR amplification Dual Flat Block GeneAmp PCR System 9700 Work Area post e QuantStudio 3D Digital PCR 20K QuantStudio 3D Digital PCR Instrument PCR room Chip reading and storage e Sealed QuantStudio 3D Digital PCR 20K Chip storage Note Ideally amplified DNA or equipment and supplies used to handle amplified DNA should not be taken out of the designated work area If the work area for amplified DNA is in a
78. io 3D Digital PCR Instrument for service transfer any experiment eds files from the instrument as explained in Transfer the results on page 66 This practice ensures that you will not lose data when the QuantStudio 3D Instrument is serviced To return your instrument for service 1 Contact your local customer care center or technical support group to obtain a copy of the Certificate of Instrument Decontamination a service notification a service call number and packaging if required Decontaminate the QuantStudio 3D Instrument IMPORTANT If the chip tray becomes contaminated with radioactivity use a commercially available decontaminant to remove the contamination If the tray cannot be decontaminated the instrument cannot be returned to Life Technologies for service Complete and sign a copy of the Certificate of Instrument Decontamination Fax the Certificate of Instrument Decontamination to the customer care center Pack the QuantStudio 3D Instrument in the provided packaging without any accessories or power cords Include a hard copy of the Certificate of Instrument Decontamination in the box Note Repairs for instruments without the Certificate of Instrument Decontamination are delayed Affix the provided postage to the box then ship the instrument to the designated facility The repair process requires 2 to 3 weeks Replace the instrument fuses TM If the fuses of your QuantStu
79. ions RS In this procedure you will prepare the PCR reactions for the samples that you intend to load to QuantStudio 3D Digital PCR 20K Chip The volumes of the example protocol have been adjusted so that two are run for each sample Table 1 PCR Reaction Mix Material renin Stock Final QuantStudio 3D Digital PCR Master Mix 2X 18 0 2X 1X TaqMan Assay 20X primer probe mix 1 8 20X 1X Diluted DNA 3 6 10 ng L 1 ng uL Water 12 6 Total volume for 1 sample 2 chips 36 0 11 Volumes include 20 excess to compensate for volume loss from pipetting 1 Remove the following from storage and allow them to warm at room temperature TM e QuantStudio 3D Digital PCR Master Mix e TaqMan Assay s 2 Review the concentration of your DNA samples and prepare dilutions if necessary 3 Gently invert the tube of Digital PCR Master Mix 10 times or gently vortex on low medium speed 4 Ina0 5 or 1 5 mL reaction tube prepare sufficient PCR reaction mix for your samples Scale the component amounts appropriately depending on the number of samples that you are running Note The following example assumes that you will load each sample to two Digital PCR 20K Chips Volume pL Material 1 sample 10 samples 2 chips 1 20 chips QuantStudio 3D Digital PCR Master Mix 2X 18 0 180 0 TaqMan Assay s 20X primer probe mix 1 8 18 0 Water 12 6 126 0
80. it 5 mL 4485813 QuantStudio 3D Digital PCR UV Kit e AAAA Batteries for UV Curing Stylus 6 e UV Activated Chip Sealant Syringe and tips e UV Curing Stylus for Chip Sealant e UV Curing Stylus Stand 4484475 QuantStudio 3D Digital PCR UV Sealant Kit e UV Activated Chip Sealant Syringe and tips 4486414 QuantStudio 3D Tilt Base for Dual Flat Block GeneAmp PCR System 9700 QuantStudio 3D Digital PCR System User Guide 91 Parts and Materials General use materials and consumables General use materials and consumables Note The following materials which are used in the preparation and use of QuantStudio 3D Digital PCR 20K Chips and the QuantStudio 3D Digital PCR Instrument can be obtained from major laboratory suppliers MLS Part ORA sri Description Source Isopropanol MLS Gloves powder free nitrile MLS Heated block capable of maintaining 40 1 C MLS Laboratory grade lint free wipes MLS Microcentrifuge MLS Pipette tips 10 to 1000 uL MLS Pipettes P10 to P1000 MLS Vortexer MLS Water DNase free sterile filtered MLS 92 QuantStudio 3D Digital PCR System User Guide Specifications and Layout Component dimensions and weights 66 c cece 93 QuantStudio 3D Digital PCR System layout and connections 94 Instrument clearances 6 eee eens 94 Electrical specificatio
81. ized for use with the Microsoft Windows 7 operating system running the following Internet browsers Computer requirements e Google Chrome v21 or later e Microsoft Internet Explorer v10 0 or later 8 QuantStudio 3D Digital PCR System User Guide About This Guide nstrument consumables Instrument consumables About the Digital The QuantStudio 3D Digital PCR 20K Chip is a 10 mm7 high density reaction plate PCR 20K Chip that consists of a single array of 20 000 reaction wells Hydrophobic coatings on the chip surface enables loading and isolation of independent PCR reactions within the reaction wells Once loaded with a PCR reaction the Digital PCR 20K Chip must be sealed using a QuantStudio 3D Digital PCR Chip Case Lid and filled with QuantStudio 12K Flex OpenArray Immersion Fluid The following illustration shows the components of the Digital PCR 20K Chip assembly Chip Case Lid The lid used to sealthe Chip 1D A label applied to the Digital PCR 20K Chip for thermal cycling QuantStudio 3D Digital PCR Chip Case and imaging on the QuantStudio 3D Lid that can be used to uniquely identify Instrument the chip to which it is applied Digital PCR 20K Chip The 10 mm2 Fill port The aperture within the Chip consumable that contains the Case Lid through which Immersion Fluid 20 000 reaction wells which suspend the is injected onto the chip individual PCR reactions for thermal O Reaction w
82. ld expel particulate material on the system components Avoid placing the instrument computer and UPS unit adjacent to heaters cooling ducts or in direct sunlight Network requirements The QuantStudio 3D Digital PCR Instrument supports IPv4 TCP IP communication and provides two methods for integrating the device into a local area network LAN The fast Ethernet adapter 10 100 Mbps with a RJ45 type connector on the rear panel of the QuantStudio 3D Instrument allows you to connect the device directly to a standard network port using a standard Category 5 Ethernet cable of the required length An optional IEEE 802 11b g n Wireless Dongle connected to the QuantStudio 3D Instrument enables a connection to compatible wireless hotspots Note See the documentation accompanying the Wireless Dongle for compatibility information The QuantStudio 3D Digital PCR Instrument requires the following if connected to a network 96 The room where the QuantStudio 3D Instrument will be deployed must contain at least one active network jack If the QuantStudio 3D Instrument will be located more than 10 feet from the network jack you must provide a shielded category 6 Ethernet cable of sufficient length If dynamic network configuration is not available a static IP address must be reserved for the QuantStudio 3D Instrument The configured DNS server must resolve internet domain names such as apps lifetechnologies com quant
83. llowing figure lists the components of the QuantStudio 3D System QuantStudio 3D Digital PCR Thermal Optional QuantStudio 3D Digital PCR Pads Chip Loader QuantStudio 3D Digital PCR Chip Q UV Activated Chip Sealant Syringe Carriers QuantStudio 12K Flex OpenArray Dual Flat Block GeneAmp PCR System Immersion Fluid 9700 QuantStudio 3D Digital PCR Master Mix QuantStudio 3D Digital PCR Instrument TaqMan Assays Not shown QuantStudio 3D Tilt Base for Dual Flat Block GeneAmp PCR QuantStudio 3D Digital PCR Sample System 9700 Loading Blade Not shown QuantStudio 3D QuantStudio 3D Digital PCR 20K Chip AnalysisSuite Software QuantStudio 3D Digital PCR Chip Case Lid 66 0 6 e680 10 QuantStudio 3D Digital PCR System User Guide Data collection Instrument filters and supported dyes Getting Started About the QuantStudio 3D Digital PCR System SAB The QuantStudio 3D Digital PCR System collects raw fluorescence data from the QuantStudio 3D Digital PCR 20K Chip following PCR amplification The instrument performs a reading of a Digital PCR 20K Chip which consists of multiple image captures in the following three phases TM 1 Excitation The QuantStudio 3D Digital PCR Instrument illuminates all wells of the Digital PCR 20K Chip within the instrument exciting the fluorophores in each reaction 2 Emission Collection The
84. lp system click JERN from any screen QuantStudio 3D Digital PCR System User Guide Troubleshooting Troubleshooting chip images using the Chip View The following table summarizes some of the more common problems that can affect imaging of Digital PCR 20K Chips Note In the examples shown below the conditions triggered a data flag in the Digital PCR 20K Chip and the conditions were diagnosed using the Chip View of the AnalysisSuite Software Observation Possible Cause Action Debris or other contaminants are No action required present on the Chip Sealant during imaging Chip view The AnalysisSuite Software can compensate for small quantities of debris or other contaminants on the Digital PCR 20K Chip QuantStudio 3D Digital PCR System User Guide 69 5 Chapter 5 Troubleshooting Troubleshooting chip images using the Chip View Observation Possible Cause Action Chip view vet A bubble was present in the Sample Loading Blade when it was used to apply the PCR reaction to the Digital PCR 20K Chip If possible use the AnalysisSuite Software to filter the low quality data points or discard the chip and run the sample again When loading the Sample Loading Blades e If you are using a manual pipette pipette to the first stop e If you are using an electronic pipette decrease your pipetting speed e Ifa bubble does form in the Sample Loading Blade
85. ly in place before proceeding UV Activated Chip Sealant Syringe Syringe packaging do not discard Chip Sealant Tip twist to attach 3 Prepare a syringe containing QuantStudio 12K Flex OpenArray Immersion Fluid a Remove the syringe plunger and tip from the packaging b Before uncapping the syringe gently pull back the plunger 1 2 mm and release it to break any resistance that may have formed during storage c Unscrew the cap from the syringe then attach the OpenArray Immersion Fluid Tip by pushing it into place IMPORTANT The application of the Immersion Fluid Tip requires force No twisting or screwing is required to attach it QuantStudio 3D Digital PCR System User Guide 35 36 me mae Prepare Samples and Load Reactions Load the Digital PCR 20K Chips d Carefully depress the plunger until Immersion Fluid flows from the tip of the assembled syringe then place it on a clean surface until ready for use IMPORTANT Confirm that the tip is locked firmly in place before proceeding IMPORTANT Use all of the Immersion Fluid within 60 minutes of uncapping the syringe Once a syringe is opened you cannot reattach the cap for later use A ___ O 1 OpenArray Immersion Fluid Syringe Immersion Fluid Tip push to attach Cap remove Lint free wipe 4 Load the PCR reaction onto a Digital PCR 20K Chip TM a Plug in and power on the QuantStudio 3D Di
86. m 23 8 cm 33 0 cm 3 2 kg Module crated 11 625 in 9 4 in 13 0 in 7 0 lbs QuantStudio 3D Digital PCR System layout and connections The QuantStudio 3D Digital PCR Instrument and the GeneAmp PCR System 9700 can be placed on separate benches or collocated as shown in the following figure If you place the units adjacent to one another provide at least 20 cm 8 inches clearance between them to allow for heat dissipation 15 2 cm 6 in 20 cm 8 in 5cm 2 in uantStudio igita nstrument ual Flat Block GeneAm stem QuantStudio 3D Digital PCR Dual Flat Block GeneAmp PCR Sy 9700 Instrument clearances The instruments require the following additional clearances IMPORTANT For safety the outlet used to power the instruments must be accessible at all times Component Top Front Sides Back m 30 48 cm 30 48 cm 15 2 cm 15 2 cm QuantStudio 3D Instrument 12 in 12 in l in Bin QuantStudio 3D Digital PCR 25 4 cm 15 2 cm 15 2 cm 25 4 cm Chip Loader 10 in 6 in 6 in 10 in Dual Flat Block GeneAmp 20 38 cm 20 38 cm 20 38 cm PCR System 9700 8 in 8 in 8 in 94 QuantStudio 3D Digital PCR System User Guide Specifications and Layout Electrical specifications Electrical specifications J WARNING For safety the power outlet used for powering the instrum
87. ment O t 78 2 In the touchscreen touch in the Main Menu 3 In the Settings Menu touch Run History to view the results for the past experiments Note The QuantStudio 3D Instrument can store up to 300 experiment files before it begins purging the files from memory When the instrument reaches the limit it replaces the oldest file in the cache with the data from the current run 4 SETTINGS Instrument Settings Data Destinations Maintenance amp Service Run History 4 In the Run History screen touch the entry of the experiment that you want to transfer or view 4 Run History Chip ID Timestamp Run Status B0498N 130610_120546 Pass BO498N 130610_102231 Pass X040098N 130609_162537 X040098N 130523 005353 QuantStudio 3D Digital PCR System User Guide Analyze the Prepared Chips irae Using the QuantStudio 3D AnalysisSuite Software S The Run History screen displays the data transfer status next to each file in the instrument memory e Pass The instrument successfully transferred the file to the selected destination e In Progress The instrument is either processing the imaging data for the associated file or transferring the file to the selected destination e Export Failed The instrument was unable to transfer the results to the selected destination e Run Failed The instrument was unable to process the imaging data for the chip 5 In the Results Details screen
88. n IP address to the instrument CHOOSE CONFIGURATION TYPE amp DHCP Static IP QuantStudio 3D Digital PCR System User Guide Appendix B Networking Connect the instrument to the network c If you elected to assign a static IP to the instrument touch the following fields and enter the e IP Address Enter the static IP address for the instrument e Netmask Enter a subnet mask for the network if applicable e Gateway Enter a default gateway for the network if applicable Note For each setting touch the associated field then enter the setting using the keypad and touch Enter to accept the value NETWORK SETUP d Touch g then configure the domain name service DNS settings e DNS Domain Enter the domain name of the DNS server that you want the instrument to use e DNS Server Enter the IP addresses for the primary top and secondary bottom DNS servers IMPORTANT DNS settings are required if you Specify the QuantStudio 3D AnalysisSuite Cloud Software as a data destination Want to use Network Time Protocol NTP for instrument date time synchronization NETWORK SETUP QuantStudio 3D Digital PCR System User Guide 85 Appendix B Networking Connect the instrument to the network e Touch g then configure the Windows Internet Naming Service WINS settings e Windows Domain Enter the domain name of the WINS server e WINS Server Enter the IP addresses for the
89. n available e Wireless connection selected connected to network e amp Wireless connection selected no connection available Data destination icon Represents the selected data destination e Cloud data destination selected e C P USB drive destination selected Destination button Lists the name of the destination to which the QuantStudio 3D Instrument will save data Touch the button to select a data destination Settings button Touch to open the Instrument Settings menu that allows you to set the system date and time the network connection and perform other maintenance and configuration tasks QuantStudio 3D Digital PCR System User Guide Chip presence indicator Indicates the presence of a Digital PCR 20K Chip within the QuantStudio 3D Instrument When a chip is loaded within the instrument the touchscreen displays the chip ID present on the QuantStudio 3D Digital PCR Chip Case Lid Note If the QuantStudio 3D Instrument cannot read the chip ID then the touchscreen displays the instrument name instead Start Run button Touch to image the loaded Digital PCR 20K Chip Note The Start Run button is inactive until a Digital PCR 20K Chip has been loaded and a viable destination is available 59 Analyze the Prepared Chips J Analyze the chips Analyze the chips Materials The following materials are required to image loaded Digital PCR 20K Chips on the required Qu
90. ng guidelines Cases e Always wear powder free gloves when handling Chip Cases IMPORTANT Never handle the Chip Cases without gloves Oils from your hands can contaminate the Digital PCR 20K Chips and interfere with imaging e Handle the loaded Digital PCR 20K Chips by gently gripping the sides of each Chip Case being careful to not apply pressure to the Digital PCR 20K Chip within it e Do not touch the surface of the QuantStudio 3D Digital PCR Chip Case Lid If you accidentally touch the surface of a Chip Case Lid clean the Chip Case using a laboratory wipe that has been thoroughly sprayed with isopropanol then dry it by wiping in one direction using a clean laboratory wipe Note Debris or excess sample on the Chip Case Lid can fluoresce and interfere with thermal uniformity e Thermal cycle loaded Chip Cases within 2 hours after loading them 9E000E0X O 52 QuantStudio 3D Digital PCR System User Guide Perform the PCR ag Thermal cycle the Digital PCR 20K Chips ae Perform the PCR Note Install the Tilt Base to the thermal cycler for the following procedure AN CAUTION PHYSICAL INJURY HAZARD During 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 IMPORTANT Thermal cycle prepared QuantStudio 3D Digital PCR 20K Chips within 2 hours after loading them IMPORTANT Thermal
91. nly onto the chip A small amount of fluid running off the surface of the chip is acceptable Do not fill the case with fluid IMPORTANT Do not allow the syringe tip to contact the Digital PCR 20K Chip 5 Rotate the Chip Loader arm so that the Chip Case Lid solidly contacts the Digital PCR 20K Chip firmly press down for 15 seconds to ensure a tight seal then press and hold the Lid Nest button while returning the Chip Loader arm to its original position IMPORTANT Press down on the Chip Loader until you reach the hard stop The arm requires gt 20 lbs of force to apply the Chip Case Lid QuantStudio 3D Digital PCR System User Guide Prepare Samples and Load Reactions Load the Digital PCR 20K Chips 6 Fill the Chip Case with Immersion Fluid a Hold the Chip Case by its edges and at a 45 angle so that air can escape from the loading port as you fill it then carefully dispense Immersion Fluid into the port until the Chip Case contains an air bubble no larger than the fill port lt 2 3 mm in diameter After filling the Immersion Fluid rotate it slightly to reveal any air bubbles that might be hidden behind the serial number label If bubbles are present add additional Immersion Fluid to remove them IMPORTANT Expel the Immersion Fluid slowly If injected too quickly the fluid can flush the reactions suspended within the chip Note Minimize air bubble creation when you dispense the fluid however
92. ns gt cerea crane erun en a p e a a a 95 Environmental requirements nases senssa rreren ene eens 95 Network requirements 0 6 66 a s a E 96 Component dimensions and weights Ensure that the installation site bench space can accommodate the system component dimensions and weight Table 2 Uncrated dimensions and weights Length Component Height depth Width Weight a 21 cm 23 3 cm 13 5 cm 2 4 kg a a a E ih 9 2 in 5 3 in 5 3 lbs QuantStudio 3D Digital PCR 11 cm 27 cm 14 cm 3 0 kg Chip Loader 4 3 in 10 6 in 5 5 in 6 1 lbs Dual Flat Block GeneAmp 26 0 cm 41 0 cm 28 0 cm 11 8 kg PCR System 9700 10 0 in 16 0 in 11 0 in 26 0 lbs assembled 1 Length increases to 38 cm 14 9 in when open Table 3 Crated dimensions and weights Length Component Height depth Width Weight QuantStudio 3D 29 5 cm 23 8 cm 33 0 cm 3 2 kg Instrument crated 11 625 in 9 4 in 13 0 in 7 0 lbs QuantStudio 3D Digital PCR 11 cm 27 cm 14 cm 3 0 kg Chip Loader crated 4 3 in 10 6 in 5 5 in 6 1 lbs QuantStudio 3D Digital PCR System User Guide 93 Specifications and Layout QuantStudio 3D Digital PCR System layout and connections Length Component Height depth Width Weight GeneAmp PCR System 26 0 cm 41 0 cm 28 0 cm 8 6 kg 9700 Base Module crated 10 0 in 16 0 in 11 0 in 19 0 lbs Dual Flat Sample Block 29 5 c
93. ntStudio 3D AnalysisSuite Cloud Software or to a Windows based or SAMBA file server for later import by the AnalysisSuite Software Note The QuantStudio 3D Instrument supports a wired network connection without any additional hardware other than what is shipped with the instrument The instrument can be configured for wireless networking through the installation of a wireless dongle which is sold separately by Life Technologies The QuantStudio 3D Instrument is designed to integrate into a standard Local Area Network LAN both wired and wireless The instrument features a Gigabit Ethernet port that provides direct connection to a network via a shielded cat 6 Ethernet cable or wireless communication via a dongle sold with the QuantStudio 3D Instrument The instrument supports the following networking technologies and standards e Communication via Transmission Control Protocol TCP over Internet Protocol IPv4 e Internet and local host name lookups via standard Domain Name Service DNS e Local server lookups via Windows Name Service WINS optional e Automatic configuration of IP DNS and WINS settings via Dynamic Host Configuration Protocol DHCP or manual configuration via the QuantStudio 3D Instrument touchscreen e Automatic data transfer to the QuantStudio 3D AnalysisSuite Cloud Software using the Hypertext Transfer Protocol over a Secure Socket Layer HTTPS or data export to a local Windows
94. ntative for assistance d When the GeneAmp PCR System 9700 displays the main menu confirm that the version number displayed on the screen is 3 12 or greater If the version number is less than 3 12 contact Life Technologies support to upgrade the instrument firmware before continuing IMPORTANT The GeneAmp PCR System 9700 must be running firmware version 3 12 or later to thermal cycle Digital PCR 20K Chips 08 00 AM 4 25 13 GeneAmp PCR System 9700 Version 3 12 ___W Y 3 12 or Name tc001 User lt lt pe gt gt better Run Create Edit Util User QuantStudio 3D Digital PCR System User Guide 23 Getting Started A Prepare the QuantStudio 3D Digital PCR System for use Bick D E fas t K Y e Program the GeneAmp PCR System 9700 with the method for thermal cycling the QuantStudio 3D Digital PCR 20K Chips e Protocol Stage Step roe uaa Ramp rate Hold ONA polymerase 96 0 10 00 activation Cycling Anneal extend 60 0 2 00 39 cycles Denature 98 0 0 30 Belaul Hold Final extension 60 0 2 00 Hold Storage 10 0 99 59 e Run speed Standard e Reaction volume 20 uL 1 Hold 2 Tmp 39 Cycles 2 Holds 96 0 98 0 Sige Method exp 001 Return Note For information on programming the GeneAmp PCR System 9700 see the GeneAmp PCR System 9700 Base Module User Manual Part no 4303481 11 Open the package containing
95. nto place then insert the plunger into the opposite end of the syringe c Place the assembled syringe within its package until ready for use IMPORTANT Confirm that the tip is locked firmly in place before proceeding lol gt A UV Activated Chip Sealant Syringe Syringe packaging do not discard Chip Sealant Tip twist to attach QuantStudio 3D Digital PCR System User Guide Prepare Samples and Load Reactions ye Load the Digital PCR 20K Chips 3 Prepare a syringe containing QuantStudio 12K Flex OpenArray Immersion Fluid a Remove the syringe plunger and tip from the packaging b Before uncapping the syringe gently pull back the plunger 1 2 mm and release it to break any resistance that may have formed during storage c Uncap the syringe then attach the OpenArray Immersion Fluid Tip by pushing it into place IMPORTANT The application of the Immersion Fluid Tip requires force No twisting or screwing is required to attach it d Carefully depress the plunger until Immersion Fluid flows from the tip of the assembled syringe then place it on a clean surface until ready for use IMPORTANT Confirm that the tip is locked firmly in place before proceeding IMPORTANT Use all of the Immersion Fluid within 60 minutes of uncapping the syringe Once a syringe is opened you cannot reattach the cap for later use S oo SSS 1 OpenArray Immersion Fluid
96. of absorbance at 260 nm and 230 nm is used as a secondary measure of nucleic acid purity The 260 230 values for pure nucleic acid are often higher than the respective 260 280 values Expected 260 230 values are commonly in the range of 2 0 2 2 If the ratio is appreciably lower than expected it may indicate the presence of contaminants that absorb at 230 nm Is intact as visualized by gel electrophoresis Has not been heated above 60 C temperatures above 60 C can cause degradation The quantity of sample added to a digital PCR reaction depends on the Concentration of genomic or complementary DNA gDNA or cDNA present in each sample Number of copies of the target sequence present in the genome or total RNA of your samples QuantStudio 3D Digital PCR System User Guide Sample dilution Determine the optimal dilution when the target is known Prepare Samples and Load Reactions JE Prepare the DNA samples Quantitation methods Before performing digital PCR experiments consider quantifying the amount of gDNA or cDNA in each sample We recommend the following methods of quantitation e Quant iT assay nucleic acid quantitation using the Qubit Quantitation Platform or e Use spectrophotometer to determine nucleic acid concentration Should a target be present at a sufficiently high concentration in the sample of interest it is possible that all reaction replicates will be positive thus preventing the digital c
97. ould use when authenticating to the AnalysisSuite Cloud Software Password Enter the password that the instrument should use when authenticating to the AnalysisSuite Cloud Software LINK TO CLOUD DESTINATION 88 QuantStudio 3D Digital PCR System User Guide Appendix B Networking Connect the instrument to the network e LAN data destination fields Target Name Enter a name to identify the LAN data destination Note The target name appears within the Destination Selection and Export screens Server Enter the path of the file server using the following syntax smb servername share path where path is optional User Name Enter the user name that the instrument should use when authenticating to the file server Password Enter the password that the instrument should use when authenticating to the file server LINK LAN DESTINATION 6 Repeat step 3 to step 5 to add additional data destinations as needed Upon saving the data destination the QuantStudio 3D Instrument automatically attempts to connect to the specified location The instrument saves the destination only if the connection is successful 7 When you are finished adding data destinations touch to return to the Main Menu QuantStudio 3D Digital PCR System User Guide 89 Parts and Materials How to order from the Life Technologies website 0 0000s eee 90 Kits consumables and accessories
98. pede pelea eee oe 72 Clean the chip tray or sample block 000 c eee eee eee eee 73 Configure the instrument settings 202 c cee eee eee eee 73 Set the instrument name ce eee teens 73 Set the date and time eri nesso etia aaeh E a eee eee eee nee 74 Returning your QuantStudio 3D Instrument for service 0c0eeeeeeeee eee 75 Replace the instrument fuses 0 00 c eect eens 75 Materials required 0 02 c cece eee eee eee 75 Replace the fuses topetea ou aes Meee ee ed RG Be ee oh whe Bes 76 Update the instrument firmware 00 cee eee eee 76 Update the firmware 000 p eia eee eens 77 View the instrument log 2 0 cece eee eee eee 77 View the log s c 8e neces ete Paes Sein aan thie bee a cele eens 78 APPENDIX B_ Networking ccceeceeeeeeeeeeeeeeeeeneeeeeees 79 Networking overview 0c e eee ee eee eee eee 79 About the network port and wireless adapter 0 2c ee eee eee eee 79 Networking guidelines and best practices cee eee eee eee eee 80 Connect the instrument to the network 2 000 e cece eee eee eee eens 80 Network setup workflow 0c e cece eee eee 80 Materials required 0 0 c cece eee eee eee 81 Required network information 0 2 0 c cece e eee eee eee 81 Connect the QuantStudio 3D Instrument to a network 00000 82 Create a netwo
99. played 3 Contact Life Technologies support Load and seal the chips using a Chip Loader 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 Wear appropriate protective eyewear and clothing IMPORTANT Wear powder free gloves while preparing QuantStudio 3D Digital PCR 20K Chips 1 Prepare the workspace a Allow the prepared digital PCR reaction to equilibrate to room temperature approximately 15 minutes b Remove the following consumables from their packaging and place them on a clean dry lint free surface TM e QuantStudio 3D Digital PCR Chip Case Lid e QuantStudio 3D Digital PCR Sample Loading Blade 34 QuantStudio 3D Digital PCR System User Guide Prepare Samples and Load Reactions j Load the Digital PCR 20K Chips CA 2 Prepare the syringe containing Chip Sealant a Remove the syringe plunger and tip from the protective packaging IMPORTANT Do not discard the protective packaging When not in use store the syringe containing Chip Sealant within the packaging b Remove the protective caps from both ends of the syringe twist the tip to lock it into place then insert the plunger into the opposite end of the syringe c Place the assembled syringe within its protective package until ready for use IMPORTANT Confirm that the tip is locked firm
100. pproximately 215 seconds remove the chip and place it on a clean dry lint free surface IMPORTANT Remove the Digital PCR 20K Chip assembly only after the ultraviolet light powers off indicating that the curing of the Chip Sealant is complete IMPORTANT Do not squeeze sealed Digital PCR 20K Chips After curing the adhesive pressure placed on the surface of the chip lid can expel the PCR reactions held within the chip wells Note If necessary you can illuminate the fill port for up to 3 minutes to ensure complete curing of the Chip Sealant 8 Visually inspect the sealed Digital PCR 20K Chip for potential problems Leaks Confirm that no Immersion Fluid is leaking from the fill port or around the seal between the Chip Case and lid Bubbles Confirm that the Chip Case is free of excessive bubbles One small air bubble is acceptable Correct lid orientation Confirm that the Chip Case Lid and the Digital PCR 20K Chip are correctly aligned If the loaded and sealed Digital PCR 20K Chip fails any of the criteria above discard it and prepare a duplicate chip Once sealed the Chip Case cannot be opened and resealed IMPORTANT Handle the Chip Cases gently being careful to not apply pressure to the Digital PCR 20K Chips within them QuantStudio 3D Digital PCR System User Guide Prepare Samples and Load Reactions Load the Digital PCR 20K Chips C Thermal cycle the prepared Digital PCR 20K Chips
101. quired for the optimal performance of the Digital PCR 20K Chips c Open the GeneAmp PCR System 9700 lid and confirm that a QuantStudio 3D Digital PCR Chip Carrier for Dual Flat Block GeneAmp PCR System 9700 is installed to both sample blocks Note Each Chip Carrier includes a set of four alignment pegs that fit into the holes of a flat sample block The Chip Carrier can fit onto either side of the sample block right or left and in any orientation 50 QuantStudio 3D Digital PCR System User Guide s Perform the PCR JE Choosing a thermal cycler 2 Power on the GeneAmp PCR System 9700 then wait for the thermal cycler to start When the GeneAmp PCR System 9700 displays the main menu confirm that the version number displayed on the screen is 3 12 or later If the GeneAmp PCR System 9700 version is less than 3 12 contact Life Technologies support to upgrade the instrument firmware before thermal cycling Digital PCR 20K Chips IMPORTANT The GeneAmp PCR System 9700 must be running firmware version 3 12 to thermal cycle Digital PCR 20K Chips 08 00 AM 4 25 13 GeneAmp PCR System 9700 Version 3 12 _ _________ 3 12 o0r Name tc001 User lt lt pe gt gt better C Run Create Edit Util User FI F2 F3 F4 FS 3 Confirm that the GeneAmp PCR System 9700 contains the method for thermal cycling QuantStudio 3D Digit
102. rk destination to receive run data 2c eee eee eee 87 APPENDIXC Parts and Materials ccceeeee scene eee eee ees 90 How to order from the Life Technologies website 0000c eee eee eens 90 Kits consumables and accessories 0 0 cece eee eee tenes 91 General use materials and consumables 0 ee eee eee cette eee eens 92 APPENDIX D Specifications and Layout ceceeeeee eens 93 Component dimensions and weights 0 cece cece eee eee eee eee 93 QuantStudio 3D Digital PCR System layout and connections 2 00 5 94 InstrumenticlearanCe eurra denenen KEE aE K ayia eae jai a ated eles eee ene be 94 Electrical specifications 0 00 a i teen eee 95 Environmental requirements 0 200 c cece eect eee eee 95 Network requirements 0 00 cece eee eee eee 96 QuantStudio 3D Digital PCR System User Guide 5 Contents APPENDIXE Safety sncccnencnsccae dsotecwr ced os aeemadenvanmaenmannasss 98 Symbols on this instrument 0 00 cece de neuk E AE O eee e eee a i 98 Conformity Symbols e632 5 2 ois deter sere beeea Thea Seach Pel lw baie eee ees 99 Safety alerts on this instrument 00 c ccc c eee eee e eet eeeeees 100 Location of safety alerts on the instrument 0 cece cece eee ee eeee 100 Safety information for instruments not manufactured by Life Technologies Corporation casera scala a a Poles hn Seeiod BE o
103. spread the reaction across the full length of the blade While filling the Sample Loading Blade e Hold the pipet so that the tip meets the sample loading port at a sharp angle and so it does not deflect either blade forward or rear Do not depress the pipette to the second stop Doing so increases the chance of introducing an air bubble to the Sample Loading Blade e Avoid creating air bubbles 44 QuantStudio 3D Digital PCR System User Guide Prepare Samples and Load Reactions Load the Digital PCR 20K Chips c While holding the Sample Loading Blade at a 70 80 angle relative to the heated block so that the port faces up place the edge of the blade at the end of the Digital PCR 20K Chip Adjust the angle of the blade until you visually confirm that it is wetting the chip Then in one smooth motion slowly drag the blade across the chip while applying a slight downward pressure to dispense the reaction IMPORTANT Move the Sample Loading Blade slowly The time required to move the blade across the chip should be approximately 10 12 seconds IMPORTANT Before you draw the blade across the chip visually confirm that the Sample Loading Blade is wetting the chip If not adjust the angle slightly A_70 80 X 40 1 C d Confirm that the Sample Loading Blade contains no residual fluid indicating that the reaction has been completely loaded onto the chip IMPORTANT Do not apply the Sample Loadin
104. ss Immersion Fluid is present on the Chip Case Lid If present remove excess Immersion Fluid from the chip lid and run the chip again If possible use the AnalysisSuite Software to filter the low quality data points Make sure to apply Immersion Fluid to each chip immediately after loading it with PCR reaction To minimize leakage when sealing each Digital PCR 20K Chip e Wear correctly fitted gloves to prevent the glove material from snagging during lid application e Make sure that the Chip Case Lid is correctly aligned to the Chip Case e Firmly press all four corners when applying the Chip Case Lid e Cure the Chip Sealant under ultraviolet light for at least 30 seconds QuantStudio 3D Digital PCR System User Guide 71 Maintenance Cleaning the chip tray and sample block 0 eee cece eee 72 Configure the instrument settings 0 c cee eee eee 73 Returning your QuantStudio 3D Instrument for service 75 Replace the instrument fuses seidr cryos erena cse iwreg ea ene 75 Update the instrument firmware 00 76 View the instrument log ces 2 een nee es 77 IMPORTANT This chapter contains all user service procedures for the QuantStudio 3D Digital PCR Instrument Procedures other than those described in this document must be performed by a qualified Life Technologies Service Engineer Cleaning the chip tray and sample block TM If a
105. studio3d and pool ntp org The names of any local file servers used as data destinations must also be available through DNS or optionally WINS QuantStudio 3D Digital PCR System User Guide Specifications and Layout Network requirements e Ifthe QuantStudio 3D Instrument will transfer experiment eds files directly to the QuantStudio 3D AnalysisSuite Software then the instrument must have outbound Internet access through port 443 HTTPS to apps lifetechnologies com quantstudio3d e Ifthe QuantStudio 3D Instrument will use Network Time Protocol NTP for date time synchronization the instrument must have outbound Internet access through port 123 UDP to the server pool at pool ntp org e Ifthe QuantStudio 3D Instrument will transfer experiment eds files to a Windows based or SAMBA file server network credentials must be provided for authentication In addition to the requirements above if the QuantStudio 3D Instrument will be connected to a wireless network the access point must support e IEEE 802 11b g n wireless standards e WEP WEP Enterprise WPA Personal or WPA2 Personal security QuantStudio 3D Digital PCR System User Guide 97 Safety A WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices
106. t 8 9 9 13 Copy View User Sort Cancel QuantStudio 3D Digital PCR System User Guide Perform the PCR ie Thermal cycle the Digital PCR 20K Chips 6 Select STD mode enter 20 uL in the Reaction Volume field then press Start F1 to begin the run Select Method Options Reaction Volume 20 pL Ramp speed MAX Enter a value from 5 to 50 pL patare 9600 MAX You can remove the Digital PCR 20K Chips from the block immediately after the final extension step at 60 C is complete and the temperature of the block is lt 30 C Sample 10 0 96 0 IMPORTANT Read the Digital PCR 20K Chips within one hour after you remove them from the GeneAmp PCR System 9700 sample block Note After thermal cycling the Digital PCR 20K Chips can remain on the sample block for up to 24 hours Note If your GeneAmp PCR System 9700 performs a 10 C hold following thermal cycling you might observe condensation on the Digital PCR 20K Chips If so use a lint free wipe to remove the moisture before imaging Unload the thermal cycler AN CAUTION PHYSICAL INJURY HAZARD During 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 Raise the heated cover lever and slide the cover back to expose the Digital PCR 20K Chips Remove the QuantStudio Thermal Pads from the sample block and s
107. t chip tray or open the GeneAmp PCR System 9700 heated cover to expose the sample block 3 If you are cleaning the GeneAmp PCR System 9700 remove the Chip Carriers and QuantStudio Thermal Pad from the sample block 4 If the contamination remains clean the chip tray or sample block using isopropanol a Pipet a small volume of 10 isopropanol solution onto surface that contacts the consumable then use a cotton swab to scrub the surface b Pipet a small volume of deionized water onto surface then use a cotton swab to scrub the surface c Use a lint free cloth to absorb the excess deionized water IMPORTANT Always use deionized water to rinse after cleaning with isopropanol solution 5 Close the heated cover or chip tray then plug in and power on the QuantStudio 3D Instrument or GeneAmp PCR System 9700 Configure the instrument settings After placing the QuantStudio 3D Digital PCR Instrument prepare it for use by configuring the system settings for your region as explained below Set the 1 From the Main Menu of the touchscreen touch then touch Instrument instrument name Settings 2 In the Instrument Settings menu touch Instrument Name QuantStudio 3D Digital PCR System User Guide 73 Sem Maintenance Configure the instrument settings 3 In the Instrument Name screen touch the Instrument Name field enter up to a 16 character name for the instrument then touch 3 to save the setting
108. tech Guest join Other Network 3 If your selected wireless hotspot is secure enter the required authentication information user name password then touch OK Q Enter Username amp Password This network requires username and password Show Pas None no network connection C Offline to configure the instrument to save data toa local USB drive The QuantStudio 3D Instrument is connected to the network when the touchscreen displays the Network Complete screen QuantStudio 3D Digital PCR System User Guide 19 Getting Started Prepare the QuantStudio 3D Digital PCR System for use e In the Network Complete screen touch OK NETWORK COMPLETE Vv You are now connected to Ethernet f In the Configuration screen touch Edit to change the instrument settings Configuration 237270218 UTC 2013 09 27 17 32 14 DHCP IP 10 43 33 12 g Inthe Edit Configuration screen modify the fields as needed then touch Save e Touch the Instrument Name field enter up to a 16 character name for the instrument then touch to save the setting IMPORTANT The instrument name is an alphanumeric string used to identify the QuantStudio 3D Instrument on the network The instrument name Can be up to 16 characters long Must consist of alphabetic and numeric characters Can contain hyphens but not as the first or last characters in the name Cannot includ
109. tem 9700 Note If not located next to the GeneAmp PCR System 9700 the QuantStudio 3D Instrument requires only 15 2 cm 6 in of clearance on either side of the instrument d Inspect the GeneAmp PCR System 9700 Base Module for damage caused during transportation If the instrument is damaged note the location and appearance of the damage then contact Life Technologies support or your service representative for assistance QuantStudio 3D Digital PCR System User Guide 21 Getting Started Prepare the QuantStudio 3D Digital PCR System for use 9 Unpack and install the Dual Flat Block Module a Open the package containing the Dual Flat Block Module b Pull the lever out from the GeneAmp PCR System 9700 Base Module Lever c Remove the Dual Flat Block Module from the package remove the protective cover place it onto the GeneAmp PCR System 9700 Base Module then push the Dual Flat Block Module back to seat the electrical connections d Push the lever into the GeneAmp PCR System 9700 Base Module to secure the Dual Flat Block Module e Inspect the Dual Flat Block Module for damage caused during transportation If the instrument is damaged note the location and appearance of the damage then contact Life Technologies support or your service representative for assistance 10 Set up the GeneAmp PCR System 9700 a Open the package containing the QuantStudio 3D T
110. ter has a network connection Note If your computer does not have a network connection contact a Life Technologies Sales Representative see Obtaining support on page 105 for information on available AnalysisSuite Software deployment options Open a browser window and go to https apps lifetechnologies com quantstudio3d Note Using your browser settings confirm that cookies and Javascript are turned on for the website to function correctly If you cannot access the website then contact Life Technologies Customer Support or email Servicedesk lifetech com Select to Launch QuantStudio 3D AnalysisSuite Cloud Software Note You can also access the software from the Software Downloads page see Obtaining support on page 105 Sign into the software using your Life Technologies user name and password or follow the instructions to create a new user account Note If you cannot sign in to the software contact Life Technologies Customer Support or email Servicedesk lifetech com The QuantStudio 3D AnalysisSuite Software features a Help system that describes how to use each feature of the user interface You can use the Help system to find topics of interest by Reviewing the table of Contents in the sidebar Searching for a specific topic Enter your search term in the Search field in the upper right then click Enter The search results will appear under the Search tab in the sidebar To access the He
111. that contains the processed imaging data and the results from the primary analysis Depending on the chosen data destination the instrument transfers the saved imaging data directly to the Life Technologies cloud for immediate access by the AnalysisSuite Software or to a network file server or local USB drive for later manual import QuantStudio 3D AnalysisSuite Software QuantStudio 3D Digital PCR System User Guide 57 Analyze the Prepared Chips Using the QuantStudio 3D Digital PCR Instrument Analysis workflow The following figure shows the workflow for running a single QuantStudio 3D Digital PCR 20K Chip on the QuantStudio 3D Digital PCR Instrument Start v Load the thermal cycled Digital PCR 20K Chip into the QuantStudio 3D Instrument y Start the run vy Review the results y Remove the Digital PCR 20K Chip y Finish 58 QuantStudio 3D Digital PCR System User Guide About the instrument interface Analyze the Prepared Chips i Using the QuantStudio 3D Digital PCR Instrument 5 The QuantStudio 3D Digital PCR Instrument features a simple interface for reading thermal cycled QuantStudio 3D Digital PCR 20K Chips Start Run Start Run Network status icon Represents the status of the QuantStudio 3D Instrument network connection e Wired Ethernet connection selected connected to network e amp Wired Ethernet connection selected no connectio
112. the hour in 24 hour format mm is the IMPORTANT If the instrument cannot minutes and ssis the seconds read the ID of a loaded chip then it uses the instrument name instead to name the file Note In the example shown above the file name indicates that the file contains data for chip BO498N which was run at 7 58 pm on May 31 2013 TM Following an image analysis the QuantStudio 3D Instrument transfers the experiment eds file for the QuantStudio 3D Digital PCR 20K Chip to the selected destination and saves a copy of the file locally to the instrument You can access and or download the experiment eds files of previous runs from the Run History screen QuantStudio 3D Digital PCR System User Guide 65 Analyze the Prepared Chips Analyzing the data Transfer the results 66 If a data transfer is unsuccessful you can use the QuantStudio 3D Instrument Run History screen to transfer experiment files to a USB drive for manual upload to the QuantStudio 3D AnalysisSuite Software Under normal conditions the instrument automatically transfers the results for each imaged chip immediately following the analysis to the cloud storage service network file server or USB drive however if the network connection fails or if the USB drive is full you can transfer the files manually as follows to make them available for secondary analysis 1 Insert a USB drive into the port on the front of the QuantStudio 3D Instru
113. the QuantStudio 3D Digital PCR Chip Loader and install to the bench where you will load QuantStudio 3D Digital PCR 20K Chips Attach the power cord to the Chip Loader and plug it onto a power outlet IMPORTANT Do not power on the Chip Loader at this time IMPORTANT Do not install the Chip Loader in an area where the enclosure may become damaged The installation of the QuantStudio 3D Digital PCR Instrument and GeneAmp PCR System 9700 is complete and the components are ready for use 24 QuantStudio 3D Digital PCR System User Guide Getting Started Prepare the QuantStudio 3D Digital PCR System for use SAD Configure the After placing the GeneAmp PCR System 9700 prepare it for use by configuring the GeneAm p PCR system settings for your region as explained below System 9700 Note This section includes the essential procedures required to prepare the settings GeneAmp PCR System 9700 for use For complete instructions see the GeneAmp PCR System 9700 Base Module User Manual Pub no 4303481 or the GeneAmp PCR System 9700 Dual Flat Block User Manual Pub no 4307808 Set the instrument date and time 1 From the Main menu press Util F4 08 00 AM 4 25 13 GeneAmp PCR System 9700 Version 3 12 Name tc001 User lt lt pe gt gt Create Edit Util User 2 From the Utilities screen press Config F3 Utilities Diag Instrument diagnostics TmCalc Calculates meltin
114. tions and instructions 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 To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of 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 with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations
115. touch Export then touch Export again to transfer the file to the USB drive lt 4 Result Details 165 1 cu 140 4 C uL 6 When finished touch several times to return to the Main Menu then remove the USB drive from the instrument After exporting experiment eds files to the USB drive you can upload them to the cloud for analysis using the QuantStudio 3D AnalysisSuite Software Using the QuantStudio 3D AnalysisSuite Software TM After the analyzed imaging data has been transferred to the QuantStudio 3D AnalysisSuite Software you can log into the software to perform the secondary analysis as described below Computer The QuantStudio 3D AnalysisSuite Software is accessed as a web based application requirements that is optimized for use with the Microsoft Windows 7 operating system running the following Internet browsers e Google Chrome v21 or later e Microsoft Internet Explorer v10 0 or later QuantStudio 3D Digital PCR System User Guide 67 Analyze the Prepared Chips fa Using the QuantStudio 3D AnalysisSuite Software Access the software from a cloud account Obtaining information from the Help system 68 When deployed in cloud configuration the AnalysisSuite Software is accessed as a web based application that can be run using any compatible web browser see Computer requirements on page 8 To access the software from a cloud account 1 Confirm that your compu
116. udio 3D Digital PCR System User Guide 41 Be Siero 42 Prepare Samples and Load Reactions Load the Digital PCR 20K Chips Load and seal the Digital PCR 20K Chips AX 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 IMPORTANT Wear powder free gloves while preparing the QuantStudio 3D Digital PCR 20K Chips 1 Prepare the workspace a Preheat the heated block to 40 C the optimal temperature for loading Digital PCR 20K Chips Note The loading surface should be a flat temperature controlled aluminum surface that is large enough to accommodate the Digital PCR 20K Chip consumable b Allow the prepared digital PCR reaction to equilibrate to room temperature approximately 15 minutes c If necessary replace the batteries of the UV Stylus in the UV Stylus Stand Note Replace the batteries of the UV Stylus after 10 hours of use 2 Prepare the syringe containing Chip Sealant a Remove the syringe plunger and tip from the protective packaging IMPORTANT Do not discard the protective packaging When not in use store the Chip Sealant syringe within the packaging and in a dark location b Remove the protective caps from both ends of the syringe twist the tip to lock it i
117. ution that you choose See Appendix B Networking for a complete description of the networking options supported by the QuantStudio 3D System TM You need the following materials to install the QuantStudio 3D Digital PCR System e Powder free gloves e Safety glasses e Scissors or pocketknife TM Before you install the QuantStudio 3D Digital PCR System do the following e Obtain the tools and materials required for installation as shown above e Confirm that the installation site meets the environmental and electrical requirements explained in Appendix D Specifications and Layout TM e Ifyou intend to connect the QuantStudio 3D Instrument to your network confirm that the installation site contains a viable connection that meets the networking requirements explained in Appendix B Networking e Read Choose additional protective devices on page 15 and if necessary obtain the additional components that you want to install TM When planning the placement of the QuantStudio 3D Digital PCR System components give special consideration to the design and organization of the laboratory To minimize the potential for contamination we recommend organizing your laboratory so that the area where amplified DNA is handled is physically isolated from the work areas for DNA extraction and PCR setup These pre and post PCR designated work areas each ideally with dedicated equipment and supplies will allow you to ma
118. work information v Connect the QuantStudio 3D Instrument to the network and power on the instrument v If dynamic network configuration is not available configure the QuantStudio 3D Instrument TCP IP settings for static IP operation v Create the network data destination s to which the QuantStudio 3D Instrument will transfer results v Test the network connection by imaging an empty QuantStudio 3D Digital PCR 20K Chip and confirming that the resulting experiment eds file appears at the specified data destination v Finish IMPORTANT The QuantStudio 3D Instrument is factory configured for DHCP operation automatic addressing network configuration is necessary only if your does not support dynamic network configuration 80 QuantStudio 3D Digital PCR System User Guide Appendix B Networking Connect the instrument to the network Materials Connection Materials required Wired CAT6 Ethernet cable of sufficient length with RJ45 connectors for a 1000Mbit s network connection or a CATS for a 100Mbit s connection Wireless SparkLAN WUBR 170GN 802 11n Single Band Wireless Dongle PN 4483658 Required network Before beginning the installation collect from a network administrator your network information policy for obtaining IP addresses DHCP or static IP If your network requires a static IP address assignment also obtain the e Static IP address for the QuantStudio
119. x in this document Revision history Revision Date Description 01 03 2013 Initial version 02 06 2013 Updated general chip preparation and instrument networking A 0 12 2013 Updated the manual chip preparation and added procedures for chip preparation using the QuantStudio 3D Digital PCR Chip Loader and wireless network installation Purpose This user guide provides step by step instructions for preparing and loading samples onto QuantStudio 3D Digital PCR 20K Chips thermal cycling the prepared consumables using a Dual Flat Block GeneAmp PCR System 9700 and analyzing the consumables using the QuantStudio 3D Digital PCR Instrument WARNING The protection provided by the equipment may be impaired if the instrument is operated outside the environment and use specifications the user provides inadequate maintenance or the equipment is used in a manner not specified by the manufacturer Life Technologies Prerequisites This guide also assumes that you have e Knowledge of techniques for handling and preparing samples for PCR e A general understanding of data storage file transfers and copying and pasting QuantStudio 3D Digital PCR System User Guide 7 About This Guide nstrument hardware Instrument hardware TM The QuantStudio 3D Digital PCR Instrument consists of the components shown in the following figure Touchscreen Provides access to the Power switch Pow
120. y open the rear panel IMPORTANT The rear panel is secured to the instrument by three pegs that require significant force to decouple If necessary insert a flathead screwdriver into the slot at the base of the panel to pry it from the instrument chassis 4 Turn the instrument over then uncap the wireless dongle and insert it into the USB port within the instrument When the device is seated within the USB port firmly push on the wireless dongle to ensure that it is plugged in completely The internal USB port is accessed through a small opening in the instrument chassis 5 Reattach the rear panel to the instrument chassis then plug in and power on the QuantStudio 3D Instrument QuantStudio 3D Digital PCR System User Guide 17 4 18 Getting Started Prepare the QuantStudio 3D Digital PCR System for use 7 Install the QuantStudio 3D Instrument a Connect the power cord to the QuantStudio 3D Instrument press the power switch to the ON position then wait for it to start about 30 sec The QuantStudio 3D Instrument is ready to configure when the touchscreen displays the End User License Agreement IMPORTANT If the QuantStudio 3D Instrument does not start contact Life Technologies support or your service representative for assistance Inthe End User License Agreement screen touch Accept to accept the agreement Inthe Network Connection screen select th

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