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ABI Prism® 7900HT Sequence Detection System

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1. Discrimination 5 z 5 N Checklist one tep escription ee Page 1 Create an allelic discrimination plate document 4 6 2a a Create detectors for the allelic discrimination probes 4 7 b Create a marker for each allelic discrimination probe 4 9 pairing c Copy the marker s to the plate document 4 10 3a Assign detector tasks to the wells of the plate document 4 11 NTC and Unknown 4 If you would like to perform thermal cycling of the allelic 4 13 discrimination plate on the 7900HT instrument create a real time plate document for the plate and program it with the the method for the allelic discrimination run Otherwise continue to step 5 5 Choose from the following If running Then a single plate continue to step 7 the first plate inaseries Save the plate document as an ABI PRISM of plates with identical SDS Template Document as explained on assay configurations page 4 17 Create a plate document from the template created in step 5 4 18 7 Configure the document with sample names and plate 4 19 information 8 a Prepare the allelic discrimination plate or plates and 4 20 perform thermal cycling on a designated thermal cycler b Run the allelic discrimination plate or plates on the 7900HT instrument 9 Analyze the run data 5 3 a Steps 2 and 3 can be eliminated
2. Center the plate 6 Again using the software to move the plate handler arm center the gripper and plate along the Y axis of the instrument tray as explained in step 4 System Maintenance 7 35 Re checking the 7 36 System Maintenance To adjust the plate handler relative to the 7900HT instrument continued Step Action 7 Using the Vertical Positioning commands carefully lower the plate handler arm onto the instrument tray and confirm that the plate rests squarely within it 8 Tighten the three black thumb screws on the platform connecting the 7900HT instrument and the plate handler 9 Release the plate from the plate handler arm a Click Open Gripper b Click Vertical Home 10 Click Find Plate The plate handler arm lowers onto the plate 11 Save the rotary and vertical offset information a Click Rotary Offset and click Yes The software records the rotary position for the plate drawer Zymark position 2 b Click Vertical Offset and click Yes The software records the vertical position for the plate drawer Now that the positions of the plate handler and instrument are fixed the plate handler Input Stack 1 stacks can be aligned and the positional values recorded To re check the position of input stack 1 Zymark position 4 Step Action 1 Place an empty plate into
3. Fixed position bar code reader cover Turn on the computer Launch the Automation Controller Software Click Open Close _Oer cose The 7900HT instrument moves the instrument tray to the out position perpendicular to the instrument From the File menu select Exit The software quits the Automation Controller Software Positioning the IMPORTANT The instrument tray must be in the OUT position to align the bar code reader Fixed Position Bar Code Reader To position the fixed position bar code reader Step Action 1 Place a plate with bar code onto the instrument tray IMPORTANT Orient the plate so that well A1 aligns to the A1 position of the instrument tray and that the bar code faces the fixed position bar code reader Well position A1 Bar code Select Start gt Programs gt PSC Laser Data gt LDHOST The LDHOST software launches and displays the LDHOST window 7 40 System Maintenance To position the fixed position bar code reader continued Step Action 3 Establish communication with the fixed position bar code reader as follows a Click the Edit button R b Click the Terminal button m The software opens the Edit Configuration and Terminal dialog boxes c From the Device Control dialog box click the Connect to Device button lt
4. Turning On the of the ABI PRISM 7900HT Sequence Detection System For a complete explanation of the 7900HT Instrument procedure see Turning on the ABI Prism 7900HT Sequence Detection System on page 3 5 To activate the ABI PRISM 7900HT Sequence Detection System Step Action Appearance 1 Turn on the monitor and computer Power buttons 2 If using an automation module turn on the Zymark Twister Microplate Handler e T B Power Press the power switch located on the IP eso dess e O switch back panel of the plate handler Rear panel of the Twister 3 Turn on the 7900HT instrument Press the power button located on the front panel of the instrument below the LED display Power Note For a description of the LED button display see Reading the Instrument Status Lights on page 3 6 4 Launch the Paora software by doing z Select one of the following i SDS 2 0 gt SDS 2 0 Select Start gt Programs gt Bh Settings or SDS 2 0 gt SDS 2 0 A Eind Help Double click the SDS 2 0 icon from F Bun io Double uliok the desktop amp Logot or AP Shut Down The computer launches the software Astar Run Setup and Basic Operation 4 5 Step 1 Creating a Plate Document About ABI PRISM Every plate run on the 7900HT instrument requires the creation of a plate document Plate Documents within the SDS software Plate documen
5. E E E E i mplitude A2 A3 A5 AB E E E E E E E E E E B B2 B3 B4 B5 B6 B8 B9 B10 B44 B12 i i E E E E E E E E E c c2 c3 c4 c5 c6 ce ca c10 ctt 42 E E E E E E E E E E D Io Fa ae ba Ra be a ne Bio fou e _ SSS Ss p D 10 20 30 i o B Wavelength E Jes E2 E3 E4 E5 E6 Temperature E E E E E E st EEEE E s F fra F2 F3 F4 F5 F6 F7 F8 F9 F40 F414 F42 40 E E E E E E E EEEE E 20 G fot 62 63 64 65 66 67 68 69 G10 611 612 E E E E E E H faa H2 H3 H4 H5 HE H7 He Ha H10 H114 H12 TE an E E 5 6 7 8 a eS a 12 Raw Data Calibration Data EEEE a a E A plitude A2 43 Aa A5 AB A7 A8 a3 Ad EEEE E E we B tet B2 B3 B4 B5 B6 B7 Bs B9 B10 B44 B12 bs E E E E E E EEE E E E C fc c2 c3 c4 c5 cB c7 cs c9 c10 ttt E E E E E EE EEE E D fo D2 D3 D4 D5 D6 D7 Ds pa D10 D411 D12 0 0 20 30 o Hi i o E i Wavelength E Jes E2 E3 E4 E5 E6 E7 ES E9 Temperature E E E EE E E E Ffa F2 F3 F4 F5 F6 F7 FS F9 40 E E E E E E 8 E 20 G Io1 62 63 64 65 668 o7 68 69 20 0 H faa H2 H3 H4 H5 HB H7 HB Ha 0 a A2 ca a z sE tH E 3H 3 sE E 3H E i 3 3E o E 3H z i D D N 30 Wavelength Temperature C 60 E E E E E E E E C fc c2 c3 c4 c5 c6 c7 cs cg c10 11 c12 E E E E E E E8 E a D f4 D2 D3 D4 D5 D6 D7 bs bg D10 D11 D12 0 E E E E E E E8 E E jes E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E E E E E E E F fra F2 F3
6. Rear Panel of the Twister If operating normally the plate handler moves the arm to the home position over the output stack Turn on the 7900HT instrument by pressing the power button located below the status lights on the front of the instrument see the figure at the top of the page If operating normally the 7900HT instrument will do the following on startup Emit a high pitched tone signalling that system has been initialized Cycle the status lights Red gt Orange gt Green indicating that the 7900HT instrument is active see Reading the Instrument Status Lights on page 3 6 for more information Getting Started 3 5 Reading the Instrument Status Lights The 7900HT instrument contains three lights located on the lower left side of the front panel to indicate the status of the instrument 3 6 Getting Started yy Ss Red Orange Status lights Green Ea EN Power button GR2010 Light Appearance Status Action Green Solid The 7900HT instrument is on None in idl a ANCU AG SAG TEACY TO TUN This state indicates normal Flashing Interlocks are open and or instrument function the scan head has not reached the safe position The instrument door is open Orange Flashing The 7900HT instrument is None transmitting receiving data l PON to from the computer This state indicates no
7. 4 34 Loading Plates onto the Automation Module aaaea anaana 4 36 Operating the 7900HT Instrument Using the Automation Controller Software 4 38 Ter NE RUM epearen Cele el ake nae Ree edna eae ee cue eke ee de 4 38 5 End Point Analysis End Point Runs on the 7900HT Instrument 0 ccc ee ees 5 2 Section Allelic Discrimination 0 0 0 0 0c ccc cee e ene e eee eeeees 5 3 OVERVIEW ais 97 Gls ate ee ra ee ee ea os Pe oe EIEE 5 4 Beror VOU CU ete 2c oo teeter a as aie ote CN en Gea Geet heen 5 7 Analysis Checklist te teh tet ba oe is ote Ae a eG ew nies alee owen Crome 5 8 Analyzing a Completed Allelic Discrimination Run 2 200000 eee 5 9 Calling and Scrutinizing Allelic Discrimination Data 0 00 e ee 5 10 ixier tHe AMAly S18 9643 2 4 64 8 ae ee ie ee a a ee oe a bau ee bees 5 15 6 Real Time Analysis Real Time Runs on the 7900HT Instrument 0 0 ee eee 6 2 Section Absolute Quantification 0 cc ccc eee eee e ee eee nnes 6 3 OVEIVIOW wrice bet iota nod cean a eae ee Mee bee El eb ees See eee 6 4 Before VOU DCC p4 5 40 50 send tay yeee eee oN aaa hee cas oS ENS RSE ee Ee aes 6 6 Analysis Checklist i Sun dus or dt eds boeken tae ete eee I Oe ee 6 7 Analyzing the R n Datars roresrreree obi Wo treated ed Ae Seas Mew hak RA es 6 8 MIC Wine ResUl een os Ait fed Bath ee aw oe ead Sarees der adenine was 6 13 Pier the ANAlVSis cv iA ooh ek cee
8. Step Action 1 Click and drag the grey line dividing the plate grid and well inspector to the right The software expands the plate grid and table pane to the new width Dividing line click and drag ay uel Pale EL GE ean a aie uy ue Setup instrument ee ee ee ee RLB GU ri M ri M rR M ri M ri M ri M nri M nR Sample Name E E E E E E E E E m RIE Ri M ri M ri M ri M Ri M ri M ri M ri M ri M ri _Use Detector ___Reporter_ Task Quantity Color _ EEEE EEHEEHE E RNaseP FAM Ol M Di el me m Di m me m me Em Di m me m Bi m met iT me m mt 2 Using the grey dividing lines resize other elements of the plate document until comfortable using the feature Exercise 2 Maximizing Minimizing Panes Views and Plots You can maximize the panes views and plots of plate documents by clicking the sizing buttons embedded within the grey dividing lines Note Sizing buttons are the arrow head marks that appear between adjacent elements of the plate document When clicked a sizing button hides the element to which it points To illustrate the maximize minimize feature maximize the plate grid as follows Step Action 1 Click the down arrow sizing button a in the divider between the plate grid and the table pane to maximize the plate grid vertically The software maximizes the plate grid by minimizing the table pane TODO Meee o G o GA N GA s G s GI s GI s Gi s
9. 0 0 00 eee eee eee B 2 Setup Table Bile POnmMaAl vindomcce blow i sheds eet hatch bedded ee db betwen orde bast B 4 ExXponing Grapnics setae Veta ans Gatto eee kaa Tk cee tee curaseeea das B 8 Exporting Plate Document Datis 24 e8 4 2 priren Se Pha Se bee cA eee es B 9 Designing TaqMan Assays Assay Development Guidelines a r 205 000 oo Sant oak GA ee eK eee es C 2 Design Tips for Allelic Discrimination Assays 0 0 0 cee eee eee eens C 5 Design Tips for Quantitative PCR Assays 00 ees C 6 Kits Reagents and Consumables References Contacting Technical Support Limited Warranty Statement Index Safety In This Chapter This chapter discusses the following topics Topic See Page Attention Words and Warning Labels 1 2 Chemical Hazards Waste Profiles and Disposal 1 3 Obtaining Material Safety Data Sheets MSDS 1 5 Safe Instrument Use 1 6 Safety 1 1 Attention Words and Warning Labels Documentation User Attention Words Instrument Safety Labels Laser Exposure Site Preparation and Safety Guide 1 2 Safety Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation 7 Mer Aol Indicates a potentially hazardous situation w
10. Allele X homozygotes End Point Analysis 5 11 5 12 End Point Analysis To call allele types continued Step Action 4 From the Call drop down list select the Allele X call Setup Instrument Results Marker CYP 2 Call fiae 7 e k W e a fs E A oe gt Select Allele X 17 0 faee y Allele X amp Y aa Efe A inn The software automatically labels the samples and wells with the Allele X call 7 Allele X homozygotes e2 aces x cna 5 Repeat steps 3 and 4 to apply calls to the rest of the samples within the plot Call Symbol Definition Allele X Homozygous for the allele displayed on the X axis of the Allelic Discrimination Plot Allele Y Homozygous for the allele displayed on the Y axis of the Allelic Discrimination Plot Both Heterozygous Alleles X and Y NTC a No Template Control Undetermined x Unknown Unlabeled Note You can adjust the appearance of the allelic discrimination plot or the data points it contains using the Display Settings dialog box See the SDS software online help for more information Setup Instrument Results Call 1 AANA AANE Allele Y homozygotes S Allele X Allele Y a heterozygotes x j Legend mel sen E l i See i Allele X homozygotes No amplification i pe x en aa i 6 If evaluating for multiple markers do the following a From the Marker drop down list sele
11. The terminal window displays the fixed position bar code reader response d Click OK to close the Information dialog box The LD Host program communicates with the bar code reader and updates the Edit Configuration dialog box with the current configuration settings Configure the software for the alignment as follows a From the bottom of the Edit Configuration dialog box locate and click the Op Modes tab Op Modes tab Note You may need to use the arrows located in the bottom of the dialog box to locate the Op Modes tab b From the Operating modes selection group of the Edit Configuration dialog box click the arrow to the right of the Mode heading and select Test from the drop down list r LD11000 Edit Configuration Ea elele Serial on line x Device Configuration On line Serial on line Automatic et t Mode drop down list c From the Device Control dialog box click RAM to toggle to EEPROM mode EN Device Control a 1X Device LD11000 Port COM4 9600 N81 Status E TX RX Commands RAM button d From the Device Control dialog box click Send e From the Confirm dialog box click YES to save to EEPROM The fixed position bar code reader begins a continuous repeating scan of the bar code The software updates the Terminal dialog box every 0 5 sec indicating the percentage of accurate reads completed during the 0 5 sec interval System Maintenance 7 41 7 42
12. 96 Well PureDye_Plate lt plate number gt _ lt date in MMDDYY format gt For example the file name for a Pure Dye Plate 1 run on May 31 2001 would be PureDye_Plate1 053101 d Click Save The software saves the plate document 9 Prepare and run the pure dye plate as explained below Preparing and IMPORTANT A background run must be performed prior to running a pure dye plate See Running the Performing a Background Run on page 7 13 for more information KUTE erate To prepare the pure dye run Step Action 1 Briefly centrifuge the pure dye plate 2 Load the pure dye plate into the 7900HT instrument as follows a From the plate document in the SDS software click the Instrument tab b From the Real Time tab in the Instrument tabbed page click Open Close The instrument tray rotates to the OUT position c Place the pure dye plate into the instrument tray Note The A1 position is located in the top left side of the instrument tray 3 Click Start The 7900HT instrument begins the pure dye run The method for a pure dye run is hard coded into the software and consists of a single 2 min hold at 60 C Note Before starting the run the instrument may pause up to 15 min to heat the heated cover to the appropriate temperature 4 When the pure dye run is complete and the Run Complete dialog box appears a Click OK to close the dialog box b Click Open Close and remove the
13. ABI PRISM 7900HT Sequence Detection System User Guide Basic Operation and Maintenance Applied Biosystems ABI PRISM 7900HT Sequence Detection System User Guide gt Applied BS Biosystems Copyright 2001 Applied Biosystems All Rights Reserved For Research Use Only Not for use in diagnostic procedures ABI PRISM and its design AmpliCover and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries ABI Applied Biosystems FAM JOE NED ROX TAMRA TET VIC and Primer Express are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries AmpErase AmpliTag AmpliTaq Gold GeneAmp SYBR and TaqMan are registered trademarks of Roche Molecular Systems Inc Windows and Microsoft are registered trademarks of Microsoft Inc Twister Universal Plate Handler is a trademark of Zymark Corporation Twister Users Guide part number 66503 from Zymark Corporation All other trademarks are the sole property of their respective owners Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Authorized Thermal Cycler This ABI PRISM 7900 HT Sequence Detection System Base Unit Serial No___ S S SC in combination with its immediately attached sample block modules com
14. File size can increase depending on the plate document s data collection options Getting Started In This Chapter This chapter discusses the following topics Topic See Page Getting Started 3 2 About This Manual 3 3 Turning on the ABI Prism 7900HT Sequence Detection System 3 5 Using the SDS Software Workspace 3 7 Basic Software Skills Tutorial 3 11 Using SDS Plate Documents 3 21 Getting Started 3 1 Getting Started Before You Begin If this is your first time using the ABI PRISM 7900HT Sequence Detection System consider completing the Basic Software Skills Tutorial on page 3 11 before continuing The tutorial will provide you with the fundamental knowledge required to operate the SDS software and will teach you time saving techniques to allow you to use it quickly and efficiently Procedure The following table contains a list of major procedures described within this manual Quick Reference Procedure See Page Turning on the ABI PRISM 7900HT Sequence Detection System 3 5 Setting Up and Running SDS Experiments Creating an SDS plate document 4 6 Running an individual SDS plate document 4 21 Running batches of SDS plate documents using the automation accessory 4 29 Stopping a run in progress From the SDS software 4 26 From the Automation Controller Software 4 38 Ejecting a plate From the SDS software 4 27 From the Automation Controller Software
15. Help button Al HD1 1 CYP 2C9 2 Allele x a A2 HD1 1 CYP 2C9 2 Allele X A3 HD1 2 CYP 2C9 2 Allele X Exporting Plots as Graphics The SDS software can export most panes and plots of the plate document as JPEG Joint Photographic Experts Group graphic files The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software To export a plot as a graphic file see Exporting Graphics on page B 8 or click the help button within the plot of interest for further instructions End Point Analysis 5 15 Real Time Analysis In This Chapter This chapter discusses the following topics Topic See Page Real Time Runs on the 7900HT Instrument 6 2 Section Absolute Quantification 6 3 Overview 6 4 Before You Begin 6 6 Analysis Checklist 6 7 Analyzing the Run Data 6 8 Viewing Results 6 13 After the Analysis 6 15 Section Dissociation Curve Analysis 6 17 Overview 6 18 Before You Begin 6 19 Analysis Checklist 6 20 Analyzing the Run Data 6 21 Determining Tm Values for the Analyzed Run 6 22 After the Analysis 6 24 Real Time Analysis 6 1 Real Time Runs on the 7900HT Instrument Real Time Runs Quantitative RT PCR 6 2 Real Time Analysis Real time is the term used to describe the category of sequence detection runs in which the ABI PRISM 7900HT Seq
16. Step Action 1 From the Detector Manager dialog box of the SDS software copy the detectors to the plate document as follows a While pressing and holding the Ctrl key click the detectors you want to apply to the plate document The software highlights the selected detectors b Click Copy to Plate Document The software adds the detectors to the well inspector of the plate document Click Done to close the Detector Manager dialog box From the plate grid select the wells containing the assay for the first detector Note For easier selection of plate grid wells use the Ctrl and Shift keys to select wells individually or in groups See page 3 16 for more information Apply detector to the selection by clicking the check box for the detector in the Use column of the well inspector Ej Untitled 1 Use check box Dete Dete Detector FAM Un known D Detector VIC Unknown o m Detector added to selected wells of the plate document Bi EP Repeat sigs 3 and 4 to apply the remaining detectors to the plate grid Configure the plate document with detector tasks as explained on page 4 11 4 8 Run Setup and Basic Operation Creating an Allelic Allelic discrimination plate documents feature the use of markers to aid in organizing Discrimination and applying detectors based on the loci they target A marker is a pairing of two Marker detectors representing ch
17. To analyze allele types continued Step Action 3 Screen for Unknown samples that failed to amplify a From the Allelic Discrimination Plot select the NTC cluster The SDS software highlights the datapoints within the allele plot and the plate grid b From the plate grid check the wells containing Unknown samples for selected wells that are clustered with the NTCs Samples that clustered with the No Template Control wells may Contain no DNA Contain PCR inhibitors Be homozygous for a sequence deletion Setup Instrument a x Legend o m ee NTC cluster selected SSO SSS cE RE RE RE RBR ERE REE Ee No Template DPR RR ERR RRR RRR eee Control wells DIDIDIDIXIDIRIDIDIDIDIDIDID Unknown samples clustered with the NTCs 4 Retest any samples that did not cluster tightly or clustered with NTCs to confirm the results 5 If evaluating for multiple markers do the following a From the Marker drop down list select a different marker b Repeat steps 1 to 4 for the new marker c Repeat steps a and b until the calls for each marker have been verified After the Analysis Changing the Plate Document Display Settings Saving the Plate Document Printing a Report Exporting Plate Document Data Before printing or exporting the analyzed data the software allows you to reconfigure the appearance of several elements of the plate document including the results table plate grid
18. sample names adding to a plate document 4 19 importing into a plate document B 2 Sample readout from the Real Time tab sample volume setting 4 15 saturation signal 7 21 saving 1 3 3 2 4 4 25 Index 6 plate documents 3 12 4 22 as template files 4 17 scrutinizing allele calls 5 13 SDS software about 2 14 disconnecting 4 28 installing 7 48 launching 3 7 learning to use the software 3 11 re connecting 4 28 upgrading 7 48 selecting wells from the plate grid 3 17 setting baseline values 6 10 threshold value 6 11 setup table file about B 4 example files B 4 exporting B 9 importing into a plate document B 2 structure B 5 signal saturation 7 21 Slope readout from the Standard Curve Plot spectral calibration See pure dye runs Stage readout from the Real Time tab Standard Curve Plot about 6 14 exporting as a graphic file B 8 exporting data as a text file B 9 standards quantifying for absolute quantification C 6 selecting for absolute quantification C 6 starting plate queue 4 38 run from the SDS software 4 24 State readout from the Real Time tab 6 14 4 25 4 25 Status readout from the Real Time tab 4 25 step adding to a method 4 15 Step readout from the Real Time tab 4 25 stopping plate queue 4 38 run from the SDS software 4 26 Support F 1 SYBR Green 1 Dye about A 3 T table pane about 3 21 exporting B 9 TaqMan fluorogenic probe about A 2 designing C
19. Calculating Threshold Cycles A 10 Theory of Operation The ABI PRISM 7900HT Sequence Detection System creates quantifiable relationships between test samples based on the number of cycles elapsed before achieving detectable levels of fluorescence Test samples containing a greater initial template number cross the detection threshold at a lower cycle than samples containing lower initial template The SDS software uses a Threshold setting to define the level of detectable fluorescence The threshold cycle C for a given amplification curve occurs at the point that the fluorescent signal grows beyond the value of the threshold setting The C represents a detection threshold for the 7900HT instrument and is dependent on two factors Starting template copy number Efficiency of DNA amplification the PCR system How the SDS Software Determines Cys To determine the C for an amplification plot the SDS software uses data collected data from a predefined range of PCR cycles called the baseline the default baseline occurs between cycles 3 and 15 First the software calculates a mathematical trend based on the baseline cycles R values to generate a baseline subtracted amplification plot of AR versus cycle number Next an algorithm searches for the point on the amplification plot at which the AR value crosses the threshold setting the default threshold setting is 0 2 The fractional cycle at which the intersection occurs is d
20. End Point Plate Documents Only Post the date that the post read was performed a A post read is a plate read performed after a plate has undergone thermal cycling Note For more information on the elements of the Real Time and Plate Read tabs click the 7 button and see the SDS software online help Run Setup and Basic Operation 4 25 Stopping the IMPORTANT Read the following directions carefully before stopping a run in progress Sequence Detector from the Run Type Action SDS Software End Point Runs From the Plate Read tab of the plate document click Stop Allelic Discrimination Real Time Runs Has the Cover reading in the Real Time tab reached 104 C Absolute NO the heated cover temperature is below 104 C Quantification From the Real Time tab of the plate document click Stop Melting Curve YES the heated cover temperature has reached 104 C The instrument has begun to run the plate and is in the process of thermal cycling Determine a course of action from the options in the following table Reason for Stopping the Run Then Forgot to make a change to the plate document such as adding a detector detector task or sample name not including mistakes to the temperature profile do not stop the run Allow the instrument complete the run then edit the plate document before analyzing the data The software does not use detector or sample information until
21. System Maintenance 7 21 To create a pure dye plate for custom dyes continued Step Action 8 Create a pure dye plate for the custom dye s by pipetting each custom dye to at least three columns of an ABI PRISM Optical Reaction Plate at the concentrations determined in step 7 i GR2107 6 IMPORTANT The optical configuration of the 7900HT instrument requires that each pure dye occupy at least 3 columns of the Pure Dye plate to permit adequate data collection Seal the plate using an optical adhesive cover or optical flat caps Create a template document for the custom pure dye plate as explained below Constructinga To create a template for running the custom pure dye plate Custom Pure Dye Plate Document Step Action Template 1 Launch the SDS software 2 Add the new dye to the software using the Dye Manager as follows a From the Tools menu select Dye Manager The Dye Manager dialog box appears b Click Add The Add Dye dialog box appears c Type a name for the custom dye and click OK The software adds the new dye to the Custom dye list d Repeat steps 2 and 3 to add any additional custom dyes to the Dye Manager e Click Done The SDS software makes the new dyes available to pure dye plate documents Create a custom pure dye plate document for the run as follows a From the File menu select New The New Document
22. Table Observation Plate handler emits grinding noise when picking up or putting down plates Plate handler arm contacts racks when retrieving or stacking plates The plate handler arm releases plates awkwardly into the plate racks Reaction plates tip or tilt when placed into the instrument tray by the plate handler arm Plate handler fails to sense or grasp plates Plates stick to the gripper fingers of the plate handler arm Plate handler does not restack plates in original locations Fixed position bar code reader not reading plate bar codes Possible Cause Recommended Action Vertical offset too low Plate detector switch set too high Plate handler rotary offset is incorrect or vertical offset is too low Re align the plate handler as explained in Aligning the Plate Handler on page 7 32 Plate sensor switch not adjusted properly Adjust the plate sensor switch as explained in Adjusting the Sensitivity of the Plate Sensor Switch on page 28 Gripper pads on the fingers of the plate handler arm are worn or dirty Change the gripper pads as explained in Cleaning and Replacing Gripper Finger Pads on page 7 43 Gripper pads are worn or dirty Change the gripper pads as explained in Cleaning and Replacing Gripper Finger Pads on page 7 43 Restack when finished option not selected Configure the Automation Controller Software to rest
23. To configure the display settings for the template Step Action 1 From the View menu of the SDS software select Display Settings The Display Settings dialog box appears 2 Configure the display settings for the Results Grid and the Results Table For more information on the Display Settings dialog box or to view the procedures for configuring the display settings for the template click the 3 button to open the SDS software online help 3 When finished click OK The SDS software applies the new display settings to the plate document 4 Go to Saving the Plate Document as a Template below IMPORTANT Saving the plate document as a template is an optional step and recommended for instances where the document can be used to create duplicate plate documents for a series of plates with identical assay configurations If you choose not to use your plate document as a template go to Step 7 Applying Sample and Plate Information on page 4 19 To save the template file Step Action 1 From the File menu of the SDS software select Save As The Save As dialog box appears From the Look in text field navigate to the Program Files gt Applied Biosystems gt SDS 2 0 gt Templates directory Note By saving the template file to the Templates directory it becomes available from the Template drop down list in the New Document dialog box From the File of type drop down list s
24. all files included with the source code and the following disclaimer 2 Redistributions in binary form must reproduce the above copyright notice this list of conditions and the following disclaimer in the documentation and or other materials provided with the distribution THIS SOFTWARE IS PROVIDED BY THE AUTHOR AND CONTRIBUTORS AS IS AND ANY EXPRESS OR IMPLIED WARRANTIES INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED IN NO EVENT SHALL THE AUTHOR OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT INDIRECT INCIDENTAL SPECIAL EXEMPLARY OR CONSEQUENTIAL DAMAGES INCLUDING BUT NOT LIMITED TO PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES LOSS OF USE DATA OR PROFITS OR BUSINESS INTERRUPTION HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT STRICT LIABILITY OR TORT INCLUDING NEGLIGENCE OR OTHERWISE ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE Contents Safety Attention Words and Warning Labels 0 0 0 c cece eens 1 2 Chemical Hazards Waste Profiles and Disposal 0 0 0 0 cee eee aes 1 3 Obtaining Material Safety Data Sheets MSDS 0 0 0 0 00 cc eee 1 5 Sate EUMENE meake Aoevke te ole ks oleae a bas Gee abedowded oes 1 6 Product Overview SYS EIKO VIE Wea er coaster eh ote read eens eee e ae hws 2 2 Section Getting to Know the Hardware ccc cen
25. and disposal AVAAN CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Safety 1 3 About Waste Profiles A waste profile was provided with this instrument and is contained in the ABI PRISM About Waste 1 4 Safety Disposal 7900HT Sequence Detection System Site Preparation and Safety Guide Waste profiles list the percentage compositions of the reagents within the waste stream at installation and the waste stream during a typical user application although this application may not be
26. consisting of a set of default steps The default temperature ramp can be customized however Applied Biosystems recommends the following guidelines to ensure the greatest separation of the derivative peaks during the analysis and therefore the maximum resolution for the Guideline Example The Start and End steps of the temperature ramp must be separated by a minimum Thermal Profile Auto Increment Ramp Rate Data Collection Stage 4 96 0 temperature difference of End step 35 C elapse 15 seconds 0 15 each Start step The ramp rate setting for the End Thermal Profile Auto Increment Ramp Rate Data Collection step of the temperature ramp must Stage 4 be 2 95 0 95 0 End step ramp rate setting TIA A data collection icon must be Thermal Profile Auto Increment Ramp Rate Data Collection placed on the temperature ramp in Stage 4 the thermal profile 95 0 95 0 Data i 5 collection icon 0 60 0 3 Go to Step 5 Saving the Plate Document as a Template on page 4 17 4 16 Run Setup and Basic Operation Step 5 Saving the Plate Document as a Template Adjusting the Display Settings Optional Saving the Plate Document as a Template Because plate documents created from the template will retain its display settings configure the display settings of the template as you would like the child plate documents to be displayed
27. do one of the following From the Instrument menu select Disconnect to discontinue communication between the software and the instrument or From the File menu select Exit to close the SDS software Do one of the following Double click the Automation Controller Software icon LF on the desktop From the Start menu select Programs gt SDS 2 0 gt Automation Controller 2 0 The Automation Controller Software launches and displays the main screen Verify that Plate Queue text field contains plate documents for all plates you intend to run To add a plate document to the plate queue see Adding Plates to the Plate Queue on page 4 35 To remove a plate document from the plate queue see Removing Plate Documents from the Plate Queue on page 4 35 JH SDS Automation Controller x File Instrument Help Plate Queue Thermal Status Plate Name C Program Files 4pplied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program Files4pplied Biosystems S C Program Files Applied Biosystems S C Program Files 4pplied Biosystems S C Program Files Applied Biosystems C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Biosystems C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Biosystems S C Program Files Applied Bios
28. operation of the instrument thermal cycling and data collection to organize and store the data gathered during the PCR and to analyze the run data The SDS software can produce the two types of plate document files described in the table below Plate Document File Extension Description ABI PRISM SDS sds SDS Single Plate Documents are the primary file you Single Plate will use They are generated for every kind of experiment and are generally used to run plates ABI PRISM SDS sdt Although optional templates are useful as Template Document time saving devices for experiments where samples are run on plates with identical assay configurations The exercises on the following pages will familiarize you with the use of SDS plate documents Getting Started 3 11 3 12 Getting Started Exercise 1 Creating a Plate Document You will need to create a plate document for every plate you run on the 7900HT instrument The following procedure explains how to create a plate document using the SDS software To create a plate document Step Action 1 If not already active launch the SDS software as explained on page 3 7 The SDS software workspace appears 2 Choose one of the following options Click the New Document button G from the General toolbar or From the File menu select New The New Document dialog appears IMPORTANT The SDS software can h
29. requirements any nonspecific amplification is not detected Basics of SYBR The SYBR Green 1 Double Stranded Binding Dye is used for the fluorescent Green Chemistry detection of double stranded DNA dsDNA generated during PCR The SYBR Green 1 Dye binds non specifically to dsDNA and generates an excitation emission profile similar to that of the FAM reporter dye When used in combination with a passive reference the SYBR Green 1 Dye can be employed to perform several SDS related experiments including quantitative PCR and dissociation cure analysis The following figure illustrates the action of the SYBR Green 1 dye during a single cycle of a PCR LUA LLIN LLL LUIN Wh Aiii LLL LIN MT An LLM LLIN ALLL A LLLI LLN ALLL ALLL When added to the reaction the SYBR Green 1 Dye binds non specifically to the hybridized dsDNA and fluoresces Dissociation O P 5 OOO EUL 3 Q O Denaturation complete the SYBR Green 1 Dye dissociates from the strand resulting in decreased fluorescence Polymerization Forward Primer 5 N 7 PERN allia all 5 j Ce TON Manni aoad Noan aooaa a C O Reverse Primer During the extensio
30. the maximum baseline is set too high Decrease the Stop baseline value by clicking and dragging the right carrot to an earlier cycle 2 000 EH 1 000 E 3 255 fid o 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 the maximum baseline is set too low Increase the Stop baseline value by clicking and dragging the right carrot to a later cycle To set the baseline and threshold values for the run continued Step Action 5 From the Plot drop down list select ARn vs Cycle The software plots the analyzed data in the graph of normalized reporter fluorescence AR versus cycle number Identify the components of the amplification curve and set the threshold so that it is Above the background Below the plateaued and linear regions of the amplification curve Within in the geometric phase of the amplification curve 2 000 E 1 1 000 E 1 Plateau phase _ Linear phase 2 166 E 1 Geometric phase Threshold setting click and drag Background j i 14 f Baseline Cycle Set the baseline and threshold for any remaining detectors present on the plate as follows a From the Detector drop down list and select another detector b Repeat steps 3 to 6 until the baseline and threshold values have been set for each detector Eliminate outliers from the analyzed run data
31. 2 to C 3 T continued TaqMan RNase P Instrument Verification Plates about 7 24 analyzing 7 26 kits D 4 preparing a plate document running 7 24 7 26 tasks See detector tasks technical support F 1 templates about 3 11 creating a single plate document 4 18 creating multiple plate documents 4 32 saving as 4 17 thermal cycler block See sample block module thermal cycling protocol See methods threshold about A 10 configuring for automatic analysis 6 8 improper setting 8 4 setting manually 6 11 threshold cycle calculation A 10 relationship to PCR product A 11 Time readout from the Real Time tab 4 25 Time Remaining readout from the Real Time tab Ta See melting temperature troubleshooting 8 2 to 8 17 7900HT Instrument 8 14 background runs 8 8 chemistry problems 8 4 to 8 7 computer 8 14 end point runs 8 13 fixed position bar code reader pure dye runs 8 10 real time runs 8 11 SDS software 8 14 Zymark Twister Microplate Handler 8 17 turning ON the ABI PRISM 7900HT Sequence Detection System 3 5 7 25 4 25 8 17 U upgrading operating system software 7 47 SDS software 7 48 user guide conventions 3 4 organization of terminology 3 3 V viewing well information 3 16 views hiding 3 16 maximizing minimizing 3 15 resizing 3 15 showing 3 16 visualizing outliers 6 12 W WARNING attention word definition 1 2 warranty statement G 1 well inspector 3 22 Y Y Inter readout from the Standard Curve Plot 6 14
32. 4 38 Analyzing Run Data Analyzing an allelic discrimination run 5 3 Analyzing an absolute quantification run 6 3 Analyzing a dissociation curve melting curve run 6 17 Maintaining the 7900HT Instrument Changing the 7900HT Plate Adapter 7 9 Replacing the Sample Block 7 4 Decontaminating the Sample Block 7 11 Performing a Background Run 7 13 Performing a Pure Dye Run 7 17 Adding Custom Dyes to the Pure Dye Set 7 21 Verifying Instrument Performance Using a TaqMan RNase P Plate 7 24 Maintaining the Automation Accessory Adjusting the Sensitivity of the Plate Sensor Switch 7 28 Aligning the Plate Handler 7 32 Aligning the Fixed Position Bar Code Reader 7 40 Cleaning and Replacing Gripper Finger Pads 7 43 a The automation accessory includes the Zymark Twister Microplate Handler and the fixed position bar code reader See Instrument Components on page 2 3 for more information 3 2 Getting Started Using the SDS The SDS software features an online help system that can guide you through the Software procedures for setting up performing and analyzing runs To get help at any time Online Help lick a Help button located within the dialog box or window in which you are working The SDS software provides two ways to access the online help as follows To Then access general help select SDS Online Help from the He
33. Allele 2 UNKN 0l O CYP 209 2 Allele 2 UNKN_ 0l OJCYP 209 2 Allele 2 UNKN_ 0l OJCYP 209 Allele 2 UNKN_ 0l Lo O anaes ems b och Oo 01 CYP 2C9 2 Allele 2 UNKN Ol CYP 20972 Allele 2 JUNKN O CYP 2C9 2 Allele 2 JUNKN D YP 2C9 2 Allele 1 YP 2C9 2 Allele 1 D1 6 CYP 2C9 2 Allele 1 Kj as mm wo co gt AD_setup table Example Setup Table File from an Absolute Quantification Run amp y 4Q_setup table xls 4 SDS Setup File Version Output Plate Size E 3 Output Plate ID RNase P sds Plated oT G Number of Detectors o o E TAMRA __ RNase Probe Example probe Pd Sewell Sample Name Detector_ Task____Ouantty SCS SSCS Be UNKN Ooo 2 UNKN aeaa M UNKN E NKN 290009000 12 17 i 0 0 00 2 A B 4 Importing and Exporting Plate Document Data About the Setup This section explains the elements of setup table files shown on the previous page Table File Format The following table describes the conventions used in the rest of this section Format Symbol Definition courier Text appearing in bolded courier font must be applied to a setup table file exactly as appears in this document Leathe Text appearing in italic courier font must be substituted with custom values when applied to a setup table file required text
34. Allelic Discrimination Data About the Allelic The SDS software graphs the results of allelic discrimination runs on a scatter plot Discrimination View contrasting reporter dye fluorescence After signal normalization and multicomponent analysis the software graphs the normalized data from each well as a single data point on the plot The following figure illustrates the components of the Allelic Discrimination plot mis Results Marker CYP 2 Call DERCE Ei Legend Allele Allele vy Allele X amp Y E HTC w Undetermined Allele Y CYP 2C419 2 1 1 0 o 0 1 0 20 3 0 40 Allele x CYP 2C19 2 2 The following table describes the elements of an SDS plate document Component Description 1 Marker drop down list Determines the marker data that the software displays within the plot 2 Call drop down list When a datapoint is selected this menu allows you to assign an allele call to the datapoint within the scatter plot 3 Toolbar Contains the following tools for manipulating the plot Icon Description k Selects individual data points by clicking or groups of datapoints by clicking and dragging a box across a group of data points Selects groups of datapoints by encircling them with the tool Repositions the view within the plot by clicking and dragging the screen eq Zooms the plot by clicking the mouse button within the pl
35. Background Container lt the appropriate plate format gt Template Blank Template 4 If the background plate is labelled with a bar code click the Barcode text field and scan the bar code number using the hand held bar code reader 5 Click OK The software creates a plate document with the attributes for a background run Note Do not modify the background plate document The method for a Background run is hard coded into the SDS software and consists of a single hold at 60 C for 2 min Because the plate contains only deionized water the plate document does not require sample or detector labels 6 Save the plate document as follows a From the File menu select Save The Save dialog appears b Click the File name text field and type Background_ lt date in MMDDYY format gt For example the file name for a background plate run on May 31 2001 would be Background_053101 c Click Save The software saves the plate document The software is now configured for the Background run 7 Prepare and run the plate as explained on page 7 15 Preparing and To conduct the background run Running a Background Plate Step Action 1 Choose from the following If using Then a background plate from a Spectral Calibration Kit Remove the plate from the freezer and allow it to thaw to room temperature an ABI PRISM Optical Reaction Plate a Pipet deionized water to each well of the plate If usin
36. Batch dialog box appears 4 Configure the Template Batch dialog box with Plate IDs as follows a Click Add Plates The Add Plates dialog box appears b Click the Plate ID text field and scan the bar code of the first plate in the batch using the hand held bar code scanner LASER HAZARD Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots Never look into the laser beam Remove jewelry and anything else that can reflect the beam into your eyes Protect others from exposure to the beam c Repeat step b for every plate in the batch d When finished click Done The plate bar codes appear within the Plate ID field 4 32 Run Setup and Basic Operation To create plate documents from a template using the Template Batch Utility continued Step Action 5 Select a destination directory to store the new plate documents as follows a Click Browse b From the Look in text field navigate to and select the directory you want to use to receive the new files c Click Open The Template Batch dialog box displays the selected destination directory in the Plate Directory text field x p Plate ID 384N0257 MO a 384N00LQQC 384NO00LR9X 384N00LQRD 384N025075 384NO0LQN 384N016N9 x Import Edit Clear All OA wh Plate Directory Destination Browse directory Create Done 6 Click Create The software creates pl
37. FR ER RLF EL EL EL EL EEL Jel eh D D D 2 D 2 2 2 2 2 2 2 2 2 2 D e e fob poh e e e e e e Jol e fol Joh e D D D D D 2 2 2 2 2 2 2 2 2 2 D e e Job fob e e e e Jol e Jol fob fol Job e D D D D D 2 2 2 2 2 2 2 2 2 2 D LoL fol pol fol fol fol fol fol fol fol JoL fol fol gE gE D D D 2 2 2 2 2 2 2 2 2 2 2 2 D gpigigigigEgEgEgEgEgEgEgEgE gE gE gE D D 2 D 2 2 2 D 2 2 2 2 2 2 2 D LoL Jol pel yoLfoLfoLfoLfoLfoL fol JoLfoL gE gE gE D D D D 2 2 2 2 2 2 2 2 2 2 2 D LJeLJeLJeLJeLJeLJeL_JeL_JelJ L_jeLleL_feLJeL_feL gE D D D 2 D 2 2 2 2 2 2 2 2 2 2 D gigigEgEgEgEgEgEgEgEGEGEgEGEGEgE D D D D D 2 2 2 2 2 2 2 2 2 2 D gigEgEgEgEgEgEgEgEgEgEGEgEgEGE gE D D D D D 2 2 D 2 2 2 2 2 2 2 D LJ LI eLJ LIJeLIJ LJeELJ LIeLJ LJeLJeLJeLJeLJeLJeL D D D D D 2 2 2 2 2 2 2 2 2 2 D SU SEN eN sR eR eR eN sR eR eR a a a a D D D 2 2 2 2 2 2 2 2 2 2 2 2 D gr gigigEgEgEgEgEgEgEgAgEgEgEgEGE 2 2 3 2 2 D D FP 2 D 2 2 2 P P e LI LJ LIELJ LJELJ LIJeLJ LJeLJeLJeLJeLJeLlel D D D D D D D D D D D D D D D D EEEE EE EEEIEE c a l a a a E E a E E aE a a E D D D D D D D D D Dd D D D D D D Exercise 1 Viewing Well Information The SDS software provides two methods for viewing the information associated with a well or wells of the plate document To view the information for a well of the plate document do one of the following Action Result Click any well in the plate grid When selected the software outlines the well in black and to select it dis
38. From the plate grid of the SDS software select the replicate wells containing the first standard in the dilution series 2 From the well inspector click the text field in the Quantity column for the appropriate detector type the number of copies of the target template the replicate wells contain and press Enter The software labels the selected standard wells with the specified quantity Ej Untitled 1 10 11 Setup Instrument A N RNaseP s RNaseP A10 A11 Sample Name fMixed E Detector Reporter Quantity Color E RNaseP fE RNase 7 RNaseP FAM Standard 1 25E3 eis bis Quantity lea I text field 3 Repeat steps 1 and 2 to configure the other sets of replicate standard wells on the plate with the appropriate quantities When finished the plate grid should contain a complete set of replicate wells labeled with the Standard task and assigned quantities which the software will use to compute the standard curve for the run 4 If necessary set the passive reference for the plate document as explained below Otherwise program the method for your run as explained on page 4 13 Setting the If using an Applied Biosystems chemistry use the default Passive Reference setting Passive Reference Applied Biosystems chemistries use the ROX passive reference molecule If running a custom chemistry select a dye to use as a passive reference for the run Note Applied Biosystems recommends using a passive refer
39. Gold DNA polymerase cleaves the probes hybridized to the matching allele sequence s present in each sample The cleavage of each matched probe separates the reporter dye from the quencher dye which results in increased fluorescence by the reporter After thermal cycling the plate is run on the 7900HT instrument which reads the fluorescence generated during the PCR amplification By quantifying and comparing the fluorescent signals using the SDS software it is possible to determine the allelic content of each sample on the plate Mismatches between a probe and target reduce the efficiency of probe hybridization Furthermore AmpliTaq Gold DNA polymerase is more likely to displace the mismatched probe than to cleave it releasing the reporter dye By running the extension phase of the PCR at the optimal annealing temperature for the probes the lower melting temperatures Tm for mismatched probes minimizes their cleavage and consequently their fluorescent contribution The figure below illustrates results from matches and mismatches between target and probe sequences in TagMan PDARs for AD assays Livak et al 1995 Livak et al 1999 W mo 7H Allele X Probe target sequence match Probe target sequence mismatch higher Ta Allele Y A AmpliTaq Gold DNA Polymerase Probe target sequence match Probe target sequence mismatch GR1556 higher Tm The table below shows the correlation between fluorescence signals and seq
40. IOOOOO00O KOOOCOOOOO CO COC000 0 OOOD000000 000000000 O O Oo O O O ae O O O O O O O O O O O OGO OOO OOO aan OCC O00 OOO O00 OOO GR2107 ABI PRism Optical 96 Well Reaction Plate O O O O Q O O O O O O O O 0000000 0 O XXXXXXI K GR2108 0 0 0 9 0 0 0 0 YY KJ KJ O O O Consumable See Appendix D Kits Reagents and Consumables for a listing of available Requirements consumables requirements and purchasing instructions 2 10 Product Overview Instrument Connections Electrical The diagram below illustrates the electrical connections between the components of Connections the ABI PRISM 7900HT Sequence Detection System 1 I p ou T o gt A D pE eecseo JJO of ry JE oto oC Jo Or J0 OOO 0 PARRA 0 Q o Communications Cable Power Cable The following table lists the communications cables Cable Type Connects To A Communication Computer Monitor Port Monitor B Comm Power Computer Mouse Port Mouse not shown C Serial Computer Serial Port
41. JOE NED ROX SYBR Green TAMRA TET VIC 3 x 96 Well Plates 4323977 Sequence Detection Systems 384 Well Spectral Calibration Kit Includes two ABI PRISM Optical 384 Well Reaction Plates one preloaded and sealed Background plate and one preloaded and sealed Spectral Calibration plate containing eight separate dye standards FAM JOE NED ROX SYBR Green TAMRA TET VIC 2 x 384 Well Plates TaqMan RNase P Instrument Verification Plates 4310982 TaqMan RNase P Instrument Verification Plate Includes one ABI PRISM Optical 96 well Reaction Plate pre loaded and sealed with Sequence Detection primers and TaqMan probe to detect and quantitate genomic copies of the human RNase P gene 1 x 96 Well Plate 4323306 TaqMan RNase P 384 Well Instrument Verification Plate Includes one ABI PRism Optical 384 well Reaction Plate preloaded and sealed with complete Sequence Detection primers and TaqMan probe to detect and quantitate genomic copies of the human RNase P gene 1 x 384 Well Plate D 4 Kits Reagents and Consumables TaqMan Pre Developed Assays and Reagents Custom Oligonucleotide Synthesis For the latest information on TaqMan PDARs covering gene expression quantification and allelic discrimination visit the TaqMan PDAR list on the Applied Biosystems web site at www appliedbiosystems com pdarlist To order custom oligonucleotides Vi
42. Module Refer to the figure below for the components discussed in this section Components l Ies i Adjustment knob Plate sensor switch E Gripper Plate stack Expansion stacks cross sectional view of the gripper Zymark Twister Microplate Handler Fixed position bar code reader Plate Stack Positions The Zymark Twister Microplate Handler alignment is performed using the Zymark Twister Software The software refers to the positions of the plate stacks differently than the Automation Controller Software The following diagram lists the positions defined by the Zymark Twister Software and the Automation Controller equivalents Zymark Twister Automation Software Controller Position 0 Output Position 1 unused Position 2 Instrument Position 3 unused Position 4 Stack 1 Position 5 Stack 2 front of instrument Bar code Position 6 Stack 3 C wet A1 Position 7 Stack 4 System Maintenance 7 27 Adjusting the Sensitivity of the Plate Sensor Switch When to Perform The plate sensor switch located underneath the arm of the Zymark Twister Microplate Handler requires adjustment under the following circumstances Materials Required Adjusting the Switch 7 28 System Maintenance When changing sample block module formats Ifthe plate handler is having difficulty sensing plates The following materials are re
43. PDF version of the MSDS For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer Safety 1 5 Safe Instrument Use Before Operating the Instrument Safe and Efficient Computer Use 1 6 Safety Ensure that everyone involved with the operation of the instrument has Received instruction in general safety practices for laboratories Received instruction in specific safety practices for the instrument Read and understood all related MSDSs y NOT NERO Avoid using this instrument in a manner not specified by Applied Biosystems Although the instrument has been designed to protect the user this protection can be impaired if the instrument is used improperly Operating the computer correctly prevents stress producing effects such as fatigue pain and strain To minimize these effects on your back legs eyes and upper extremities neck shoulder arms wrists hands and fingers design your workstation to promote neutral or relaxed working positions This includes working in an environment where heating air conditioning ventilation and lighting are set correctly See the guidelines below MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by the following potential risk factors which include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation
44. Sample Name lt tab gt Detector lt tab gt Task lt tab gt Quantity lt tab gt Detector lt tab gt Task lt tab gt Quantity lt cr gt Example Well Sample Name Detector Task Quantity DetectorTask Quantity 8 Well This section defines the contents of the plate wells The setup table file must contain a Definition definition for each well used on the plate Each well definition list consists of one string of List characters terminated by a lt cr gt The definition can be of three main functional divisions Well number The first tab delimited text block defines the number of the well on the plate Well numbers start at 1 for well A 1 upper left corner of the plate and increases from left to right and from top to bottom The wells must be listed in order 1 2 3 Sample name The second tab delimited text block defines the name of the sample assigned to the well Detector assignments The remaining tab delimited text blocks for the well definition define the detectors assigned to the well Each detector is represented by three text blocks that define the following information The name of the detector The task assignment of the detector for the well UNKN Unknown STND Standard NTC No Template Control The quantity assignment of the detector for the well For wells containing standards assign the quantity for the standard sample in initial copy number For all other wells ass
45. The background component is a composite signal found in all spectral data that consists of fluorescence from several sources including the background electronic signal the sample block the water within the consumable and the plastic consumable itself Because the background signal can interfere with the precision of SDS data the 7900HT instrument has been engineered to minimize the background signal Additionally the SDS software also algorithmically eliminates the background signal from each fluorescent sample to maximize the instrument s sensitivity see page A 5 The following materials are required to perform a background run Material Part Number One of the following Background Plate included in a ABI PRISM Sequence Detection Systems Spectral Calibration Kit or Make a background plate requires the following See page D 4 ABI PRISM Optical 394 or 96 Well Reaction Plate ABI PRISM Optical Adhesive Cover or ABI PRISM Optical Flat Caps Pipettor 100 uL with pipet tips Centrifuge with plate adapter System Maintenance 7 13 Prepare a To prepare a plate document for the background run Background Plate Document 7 14 System Maintenance Step Action 1 Launch the SDS software 2 From the File menu select New The New Document dialog appears 3 Configure the New Document dialog box as follows Drop Down List Select Assay
46. and plate 4 19 information J 8 Prepare and run the absolute quantification plate or plates 4 20 9 Configure the analysis options for the run 6 8 10 Analyze the run data 6 9 11 Set the baseline and threshold values for each detector 6 10 12 Visualize outliers and eliminate any outlying amplification 6 12 from the run data 13 View the results of the absolute quantification run 6 13 14 Choose from the following post analysis options 6 15 Reanalyze the run data Adjust the display settings for the results table plate grid and plate document plots Print elements of the plate document Export the plate document results table or plots a Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab delimited text file See Importing Plate Document Setup Table Files on page B 2 for more information Real Time Analysis 6 7 Analyzing the Run Data 6 8 Configuring the Real Time Analysis Before analyzing the data collected from the complete absolute quantification run Analysis Options decide whether to configure the options for the software analysis The analysis Optional Options allow you to pre configure the threshold and baseline settings applied to the plate document data during the analysis If you choose not to configure the analysis options the SDS software uses the default baseline range and automatically calculates the threshold value for the
47. at the same location as their group but diverge slightly at other wavelengths c Click Next to view the next three wells d Repeat steps a to c for all remaining wells until prompted with a message reporting the extraction of the pure dyes The software extracts the pure spectra and stores the data as a component of the calibration file 3 From the File menu select Save The software saves the plate document 4 From the File menu select Close The software closes the plate document 5 If performing spectral calibration of a 96 well block module repeat the previous procedures on pages 7 18 7 19 and 7 20 to run the second Pure Dye plate Adding Custom Dyes to the Pure Dye Set When to Perform The ABI PRISM 7900HT Sequence Detection System can be used to run assays designed with custom dyes dyes not manufactured by Applied Biosystems Materials Required Creating a Custom Pure Dye Plate However before using custom dyes with the 7900HT instrument you must create and run a custom pure dye plate The following materials are required to perform a pure dye run Material Part Number ABI PRISM Optical 394 or 96 Well Reaction Plate ABI PRISM Optical Adhesive Cover or ABI PRISM Optical Flat Caps See page D 3 Custom Dye s Pipettor 100 uL with pipet tips Centrifuge with plate adaptor a Used to add custom dyes to the Dye Set Note The purpose of the cus
48. be approximately the same as the distance from the user s eyes to the keyboard For most people the reading distance that is the most comfortable is approximately 20 inches The workstation surface should have a minimum depth of 36 inches to accommodate distance adjustment Adjust the screen angle to minimize reflection and glare and avoid highly reflective surfaces for the workstation Use a well designed copy holder adjustable horizontally and vertically that allows referenced hard copy material to be placed at the same viewing distance as the screen and keyboard Keep wires and cables out of the way of users and passersby Choose a workstation that has a surface large enough for other tasks and that provides sufficient legroom for adequate movement Safety 1 7 Product Overview In This Chapter This chapter discusses the following topics Topic See Page System Overview 2 2 Section Getting to Know the Hardware 2 3 7900HT Instrument 2 4 Computer 2 7 Bar Code Readers 2 8 Zymark Twister Microplate Handler 2 9 Compatible Consumables 2 10 Instrument Connections 2 11 Section Getting to Know the Software 2 13 Sequence Detection System Software Components and Features 2 14 Managing Sequence Detection System Data 2 15 Working with SDS Software Files 2 16 Product Overview 2 1 System Overview About the 7900HT Instrument Supported Runs and Chemistries 2 2 Produ
49. be substituted for water in the third treatment Remove any remaining isopropanol or water from the wells of the sample block module Replace the sample block as explained in Replacing the Sample Block on page 7 7 10 Run a background plate to confirm that the contamination has been removed Performing a Background Run When to Perform Purpose of Background Runs About the Background Component Materials Required Applied Biosystems recommends running a background plate weekly or as often as necessary depending on instrument use A background run measures the ambient fluorescence in a background plate containing deionized water During the run the 7900HT instrument conducts a continuous scan of the plate for 2 minutes at 60 C Afterwards the SDS software averages the spectrum recorded during the run and is used to extract the resulting spectral component to a calibration file The software uses the calibration file during subsequent runs to remove the background signal from the run data Because the background signal can change with instrument age Applied Biosystems recommends regenerating the Background component calibration every month Note Background runs can also be used to detect and troubleshoot sample block contamination Fluorescence collected by the ABI PRISM 7900HT Sequence Detection System includes a background component a fluorescent signal that is inherent to the system
50. box Select the wells containing the assays for a marker you configured in the previous procedure Note For easier selection of plate grid wells use the Ctrl and Shift keys to select wells individually or in groups See page 3 16 for more information From the marker inspector click the Use check box of the marker you want to add to the selected wells The software labels the selected wells with the marker and its detectors Note The detectors associated with the marker are automatically applied to the selected wells when the marker is placed in Use Ej Untitled 1 Setup Instrument 1 2 i A Bj Anere 2 i Anei 2 Dy aticte 1 E aticte 1 Sample Name Mixed B i m Use Detector Reporter Task Quantity Color Iv Allele 1 FAM Unkno ET v Allele 2 IC Unkno Dy Anere 2 PJ Anere 2 C Allele 1 FAM Unkno Py Aliete 4 E allele 4 Allele 2 IC Unkno Cc at E Add Passive Reference D eee emove Rox of 5 fts 2 PJ Anere 2 Locus Color E Anere 1 E Anere 1 M PDAR CYP 2C9 2 i PDAR CYP 2C19 2 ay EF Detectors associated with the PDAR CYP 2C9 2 marker oO oc oO o Use check box for the PDAR CYP 2C9 2 marker selected If necessary repeat steps 3 and 4 to assign additional markers to the plate document Configure the plate document with detector tasks as explained in Step 3 Configuring the Plate Document with Detector Tasks on pag
51. complete 4 27 4 24 Run Setup and Basic Operation Operating the 7900HT Instrument Using the SDS Software Monitoring The SDS software displays instrument status and run progress in the Real Time tab Instrument Progress real time runs or the Plate Read tab end point runs of the respective plate document The following figure shows examples of the tabs during operation of the 7900HT instrument Real Time Queue Plate Read Queue Status Method Running Time Remaining 00 01 Status Method Running Temperature Time Remaining 00 01 Sample 25 0 Block 24 9 Cover 89 5 Data Collection Stamp Post Cycle Stage 1 Step 1 Time 00 00 Rep 1 State Not Started Real Time Tab Plate Read Tab Real Time Runs End Point Runs Report Displays Status the condition of the 7900HT instrument Time Remaining the calculated time remaining in the run Temperature group box Real Time Plate Documents Only Block the actual temperature of the sample block module Cover the actual temperature of the heated cover Sample the calculated temperature of the samples Cycle group box Real Time Plate Documents Only Rep the current cycle repetition Stage the current stage of the thermal cycling State the current condition of the cycle stage Step the current step being run Time the calculated time remaining in the current step Data Collection Stamp group box
52. dialog box appears c Confirm that the software has closed by looking for the Zymark Twister Software entry in the Task list If the software is still running click the software entry and click Close to exit the remaining software d From the File menu select Exit System Maintenance 7 31 Aligning the Plate Handler When to Perform Perform the following procedure if the ABI PRISM 7900HT Sequence Detection System is moved or the Zymark Twister Microplate Handler becomes mis aligned Symptoms that the plate handler is out of alignment include Excessive downward movement of the plate handler arm the arm grinds when grasping or releasing plates The plate handler arm collides with the plate stacks The plate handler arm releases plates above the bottom of the plate stacks Reaction plates tip or tilt when placed into the instrument tray by the arm Preparing the To move the instrument tray to the OUT position Instrument for the Alignment 7 32 System Maintenance Step Action 1 Remove the covers for the fixed position bar code
53. dialog box appears b Configure the drop down lists with the following options Drop Down List Select Assay Pure Dyes Container lt the appropriate plate format gt Template Blank Template c Click OK The software creates a new plate document 7 22 System Maintenance To create a template for running the custom pure dye plate continued Step Action 4 Apply pure dyes to the custom plate document as follows a Select the wells containing the custom dye b From the Setup tabbed page click the Dyes drop down list and select the appropriate dye The software applies the dye to the selected wells c Repeat steps 6 and 7 to configure the plate document with any additional custom dyes Setup instrument Dyes Custom 01 Dyes drop down list __ Custom dye added to selected wells of the plate document oi 5 Save the custom pure dye plate document as a template file as follows a From the File menu select Save The Save dialog appears b Navigate to Program Files gt Applied Biosystems gt SDS 2 0 gt Templates The Templates directory appears within the Look in text field By saving the template to the Templates folder it becomes available from the Template drop down list in the New Document dialog box c From the Files of type drop down list select ABI PRisM SDS Template Document d Click the File name text field and type a name
54. discrimination the throughput of the Throughput for ABI PRISM 7900HT Sequence Detection System can be increased by dividing the End Point Runs Workload between the 7900HT instrument and several thermal cyclers Unlike real time runs the 7900HT instrument collects data for end point runs after the completion of the PCR Consequently the thermal cycling of end point plates can be done elsewhere and then transferred to the 7900HT instrument afterwards for data collection and analysis IMPORTANT To perform the thermal cycling and the plate read using the 7900HT instrument run the plate first as a real time plate document and then again as an allelic discrimination plate document see Step 4 Programming the Plate Document Method on page 4 13 for the procedure 4 2 Run Setup and Basic Operation Setup Checklists Experiments Runs_ See the appropriate page for the type of experiment or run you want to perform Performed on the 7900HT Instrument ExPeriment Run See Page Absolute Quantification 4 3 Allelic Discrimination 4 4 Background 7 13 Dissociation Curve Melting Curve 4 4 Pure Dye Spectral Calibration 7 17 RNase P Instrument Performance Verification 7 24 Absolute To create and set up a plate document for an absolute quantification run Quantification 5 z E 5 Checklist one tep escription See Page 1 Create an absolute quantification
55. discrimination C 2 to C 5 attention words definitions 1 2 automation accessory components 7 27 fixed position bar code reader See fixed position bar code reader plate handler See plate handler Index 1 A continued Automation Controller software about 2 14 4 34 adding plates to the plate queue 4 35 configuring for operation 4 37 ejecting aplate 4 38 launching 4 34 monitoring instrument progress 4 38 removing plates from the plate queue 4 35 starting the plate queue 4 38 stopping the plate queue 4 38 B background run 7 13 to 7 16 about 7 13 constructing a background plate 7 15 creating a plate document 7 14 extracting 7 16 preparing a background plate 7 15 troubleshooting 8 8 when to perform 7 13 bar code information entering into a plate document 3 19 4 19 bar code readers fixed position See fixed position bar code reader hand held See hand held bar code reader baseline about A 10 configuring value for automatic analysis 6 8 setting manually 6 10 beam splitter See instrument optics system Block readout from the Real Time tab 4 25 bold text convention 3 4 C calibrating the 7900HT instrument adjusting the plate sensor switch 7 28 aligning the fixed position bar code reader 7 40 aligning the plate handler 7 32 performing background run 7 13 performing pure dye spectral run 7 17 CAUTION attention word definition 1 2 changing gripper finger pads 7 43 pane view and plot sizes 3 15 sample block module 7 4
56. environmental factors Use a seating position that provides the optimum combination of comfort accessibility to the keyboard and freedom from fatigue causing stresses and pressures The bulk of the person s weight should be supported by the buttocks not the thighs Feet should be flat on the floor and the weight of the legs should be supported by the floor not the thighs Lumbar support should be provided to maintain the proper concave curve of the spine Place the keyboard on a surface that provides The proper height to position the forearms horizontally and upper arms vertically Support for the forearms and hands to avoid muscle fatigue in the upper arms Position the viewing screen to the height that allows normal body and head posture This height depends upon the physical proportions of the user Adjust vision factors to optimize comfort and efficiency by Adjusting screen variables such as brightness contrast and color to suit personal preferences and ambient lighting Positioning the screen to minimize reflections from ambient light sources Positioning the screen at a distance that takes into account user variables such as nearsightedness farsightedness astigmatism and the effects of corrective lenses When considering the user s distance from the screen the following are useful guidelines The distance from the user s eyes to the viewing screen should
57. hard drive SDS files can be archived using a data compression utility The compression utility archives files by encoding them ina compressed form thereby reducing the size of a file SDS files can be compressed and decompressed many times several commercially available compression utilities are available PKZIP and arc are archive formats common to the Microsoft Windows operating system Defragmenting the Hard Drive Upgrading the Operating System Software Applied Biosystems recommends defragmenting the hard drive of the computer attached to the instrument at least once every week or before fragmentation reaches 10 As the ABI PRISM 7900HT Sequence Detection System is used and files are deleted and created the free space on the computer hard drive eventually is split into increasingly smaller blocks called clusters Consequently as the SDS software creates new files and extends old ones the computer cannot store each file in a single block Instead the system will fragment the files by scattering their component pieces across different sectors of the hard drive The fragmentation of SDS files decreases the performance of both the SDS software and the computer operating system As the hard drive becomes fragmented programs take greater time to access files because they must perform multiple seek operations to access the fragments Several commercially available software utilities are available for repairing fragme
58. in the range of 58 60 C using the Primer Express software Limit the G and or C bases on the 3 end The five nucleotides at the 3 end should have no more than two G and or C bases Place the forward and reverse primers as close as possible to the probe without overlapping the it Use an annealing temperature of 60 C for quantitative PCR and 62 C for allelic discrimination except for TagMan PDARs for Allelic Discrimination Designing TaqMan Assays C 3 Order Reagents Quantitate the Probes and Primers Prepare Master Mix Optimize Primer Probe Concentrations Run Your Custom Assay Note Because part numbers can change as new and improved products are introduced Contact your Applied Biosystems sales representative for specific ordering information You will need the following reagents and equipment to create your own applications Custom Synthesized TaqMan Probes Sequence Detection Primers TaqMan Universal PCR Master Mix optimized for TaqMan reactions containing AmpliTaq Gold DNA Polymerase AmpErase UNG dNTPs with dUTP ROX Passive Reference l and optimized buffer components IMPORTANT PCR master mix used with the 7900HT instrument must contain a passive reference dye The SDS software uses the signal from the passive reference to normalize the reporter fluorescence making well to well comparisons possible All Applied Biosystems master mix products contain an optimal concentration
59. input stack 1 2 From the Zymark Twister Software click the icon for stack 4 The plate handler arm moves over the input stack Using the Vertical Positioning commands lower the plate handler arm until itis 1 cm above the stack and verify that it is centered on the stack If necessary center the stack using the Rotary Adjustment arrows Carefully lower the plate handler arm into the stack Center the gripper as it moves down the stack by adjusting the Rotary Adjustment arrows if needed Once the plate handler arm is centered within the stack click Find Plate The plate handler arm lowers upon the plate Confirm the following The plate is in the middle of the gripper span The plate sensor switch is contacting the plate The gripper does not contact the side of the stack Click Close Gripper Click Vertical Home The plate handler raises the arm to its highest position If the plate contacts the sides of the stack re adjust the rotary position of the plate handler arm until the plate moves freely within the stack Note Contact between the plate and the stack or all stacks may be unavoidable However try to minimize the contact as much as possible To re check the position of input stack 1 Zymark position 4 continued Step Action 8 Click Rotary Offset and click Yes The software re records the rotary position for the input stack 1 Zymark position 4 9 Wh
60. module See Page supports multiple consumable formats 2 10 provides several different modes of operation including 9600 mode and 4 13 programmable temperature ramps reduces instrument downtime by allowing immediate replacement of the block 7 4 permits easy access to the sample block for troubleshooting and maintenance Product Overview 2 5 Optics System IMPORTANT Do not remove the cover to the 7900HT instrument Only a qualified Applied Biosystems service engineer may repair or adjust the internal components The optical system of the 7900HT instrument is based on the optics system found in the ABI PRISM 7700 Sequence Detection System The figure below illustrates the components of the 7900HT optics system Note For more information about the operation of the optical system of the 7900HT instrument see Appendix A Theory of Operation Charged coupled device array Camera lens Cs Grating aes gt Emission filter E 1 Laser source Beam splitter i ss Fresnel Lens within Lensplate 384 96 Well ait optical plate ANY WANNA as Side View Front View 2 6 Product Overview Computer Description System Requirements Hard Drive Partitions The computer coordinates the operation of the Sequence Detection instrument automation module and the bar code readers via the SDS software T
61. of the Passive Reference I ABI Prism Optical Reaction Plates ABI Prism Optical Adhesive Covers or ABI PRISM Optical Flat Cap Strips TaqMan Spectral Calibration Reagents Centrifuge with 384 or 96 well plate adapter Deionized water or Tris EDTA buffer 10 mM Tris HCl 1 mM EDTA pH 8 0 Disposable Gloves gt gt gt gt gt gt Use a spectrophotometric method to determine the concentrations of the probes and primers received See the TaqMan Universal PCR Master Mix Protocol P N 4304449 for specific information about primer and probe quantification Refer to the TaqMan Universal PCR Master Mix Protocol P N 4304449 for specific information about preparing the master mix for use IMPORTANT PCR master mix used with the 7900HT instrument must contain a passive reference dye The SDS software uses the signal from the passive reference to normalize the reporter fluorescence making well to well comparisons possible All Applied Biosystems master mix products contain an optimal concentration of the ROX passive reference dye Note Applied Biosystems protocols are available on the Applied Biosystems Company Web Site see Appendix F Contacting Technical Support for more information Refer to the TaqMan Universal PCR Master Mix Protocol P N 4304449 for specific information about preparing the master mix for use Note Applied Biosystems protocols are available on the Applied Biosystems Company Web Site see Appendix F
62. page 3 10 for more information Getting Started 3 7 About the All software operations and displayed information occur within the workspace of the Software Interface SDS software The workspace provides quick access to all elements of the software through the menubar and a pair of toolbars The following figure summarizes the features of the user interface of the SDS software 0 x Om File Edit View Tools Instrument Analysis Windows Help gt Caf a a a em t C 3 The following table describes the elements of the workspace Number Component Description 1 Menubar Contains a directory of menus that govern the operation of the software 2 General Toolbar Contains clickable icons for controlling the basic functions of the software file management and basic editorial 3 Display Toolbar Contains clickable icons for controlling the display of information within the SDS software workspace 4 Workspace Contains all plate documents dialog boxes and message boxes used by the SDS software 5 Message Bar Displays a variety of messages to indicate the status of the instrument Note See About the Status Bar on page 3 10 fora complete description of the message bar 6 Instrument Indicates the status of the connection to the 7900HT Connection Icon instrument Note See About the Status Bar on page 3 10 fora complete description of the instrument connection icon 3 8 Getti
63. page 4 7 for information on applying detectors to the plate document Task The task NTC Standard or Unknown assigned to the well Note See page 4 11 for information on applying detector tasks to the plate document Cr The threshold cycle generated by the well during the PCR Quantity For wells containing Unknown samples this column displays the starting copy number for the well calculated by the software from the standard curve For wells containing Standard this column displays the quantity assigned to the well Note See page 4 11 for information on applying quantities to standard wells of the plate document The following two columns contain data only if a well is run as part of a replicate group Qty Mean The arithmetic mean for the quantity values of the replicate group associated with the well Qty stddev The standard deviation of the quantity values of the replicate group associated with the well Real Time Analysis 6 13 Viewing the The software displays the standard curve generated from the run data within the Standard Curve Results tab of the plate document The standard curve plot displays the unknown samples on a graph of C threshold cycle vs initial quantity LogN The following figure illustrates the components of the standard curve plot Setup Instrument Results Help button Standard Curve Plot Detector RNase P gt Detector dro
64. plate the clamp applies a downward pressure of 70 lbs 31 8 kg onto the consumable During the run the clamp maintains a constant temperature of 105 C 3 C to prevent condensation within the consumable wells Interchangeable P ALGUIN PHYSICAL INJURY HAZARD Hot Surface Use care when working around Thermal Cycler this area to avoid being burned by hot components Blocks The 7900HT instrument features a Peltier based interchangeable sample block module based on the technology established in the GeneAmp PCR System 9700 thermal cycler The internal Peltier heating cooling unit is housed in the sample block module The sample block module is made of aluminum to provide the optimal thermal transfer rate A __ i Circuitry and connections o to the instrument BIS Do Not Touch Heat sinks Do Not Touch Top View Bottom View The sample block module provides Wide temperature range 4 99 9 C Accuracy 0 25 C from 35 99 9 C Heat cool rate 1 5 C per second Temperature uniformity 0 5 C measured 30 sec after the clock starts gt gt gt gt Long term stability and high reliability The interchangeable sample block
65. plates darkens the wells absorbing light The sample blocks should be periodically inspected for cleanliness Sample block contamination can be visualized by running a background plate and inspecting the resulting background signal for aberrant peaks above 2500 FSU see page 7 13 See page 7 11 for instructions on decontaminating the sample block Only ABI PRISM optical grade reaction plates optical adhesive covers and ABI PRISM optical flat caps should be used with the ABI PRISM 7900HT Sequence Detection System The plastics that comprise the optical parts undergo special testing for the absence of fluorescent impurities Optical reaction plates are frosted to improve the degree and precision of light reflection Bent creased or damaged plastics may adversely affect the transmission of fluorescent signal or prevent proper sealing of a well resulting in evaporation change in sample volume and altered PCR chemistry Make sure to use the correct plastics and visually inspect each reaction plate before use Note See Appendix D Kits Reagents and Consumables for a list of compatible consumables and reagenis When amplifying samples that contain very low quantities of nucleic acid generally less than 100 molecules expect lowered precision due to the Poisson distribution and biochemical effects related to binding probabilities Low copy templates are also more susceptible to losses due to non specific adhesion to plastic wells pip
66. should be avoided Position the polymorphic site in the central third of the probe Note The polymorphic site can be shifted toward the 3 end to meet the above guidelines however the site must be located more than two nucleotides upstream from the 3 terminus The following figure illustrates the placement of a polymorphism in an example probe N Nucleotide First try to position the polymorphic Do not place site in the central third of the probe it here _ 5 3 NNNNNNNNNNNNNNINNNNNNN Polymorphism If necessary place the polymorphism here Designing TaqMan Assays C 5 Design Tips for Quantitative PCR Assays Selecting an Selecting a good amplicon site ensures amplification of the target mRNA without Amplicon Site for o amplifying the genomic sequence pseudogenes and related genes Gene Expression Applied Biosystems recommends the following guidelines when selecting an amplicon Assays site for quantification assays Primers and probes must be designed following the Assay Development Guidelines on page C 2 The amplicon should span one or more introns to avoid amplification of the target gene in genomic DNA The primer pair has to be specific to the target gene and does not amplify pseudogenes or other related genes Test amplicons and select those that have the highest signal to noise ratio such as those yielding low C s with cDNA and no amplification wi
67. task from the drop down list The SDS software labels all selected wells with the task Setup instrument Sample Name Mixed Quantity 1 2569 Detector RNaseP Reporter Task drop down list 3 Repeat steps 1 and 2 to apply any remaining tasks to the plate document Choose from the following options If constructing a plate document for Then absolute quantification assign quantities to the standard wells of the plate document as explained on page 4 12 IMPORTANT The software will disregard data from all Standard wells that have Quantity values of 0 allelic discrimination if necessary set the passive reference for the plate document as explained page 4 12 Otherwise continue to Step 4 Programming the Plate Document Method on page 4 13 Run Setup and Basic Operation 4 11 Assigning Quantities For the SDS software to create a standard curve for quantification of unknown to Standards for Samples absolute quantification plate documents must contain quantity values for the Absolute Standards contained on the plate The software expresses quantity as the number of Quantification copies of the target sequence present within an individual well of the plate IMPORTANT The SDS software excludes from analysis data from Standard wells assigned a Quantity value of 0 To apply quantities to the plate document Step Action 1
68. the Fixed Position Bar Code Reader 00 c cece eee eee eee 7 40 Cleaning and Replacing Gripper Finger Pads 0 0c ccc eee eee eee 7 43 Section Maintaining the Computer and SDS Software 0000000s 7 45 General Computer Maintenance 0 0 cc eee eens 7 46 Maintamine the SDS Softwares lt 6 ckicdn eho Mee ne ee aes we ewe heehee dad 7 48 8 Troubleshooting Troubles OG tine TaD 2 sos wen oie win 4 eyed we Oe Ae eed Ce ds ede tee rer on 8 2 Low Precision or Irreproducibility 20 0 0c ccc ccc ce eens 8 4 Backo rownd RUNS earras es eK Ge AEE ESO EAS RAs Redes hbase Seow ee 8 8 Pure Dye RODS roa 8 Sk aha Sate ete eee Se Keak eee neo aac n 8 10 Real Time Runs Quantitative PCR and Dissociation Curves 05 8 11 End Point Runs Allelic Discrimination 0 0 0 0 0 cc cece eee ens 8 13 Soltware and 7900H T Instrument r eerri hirere reinn eai ena a a A 8 14 Zymark Twister Microplate Handler and Fixed Position Bar Code Reader 8 17 9 User Bulletins Theory of Operation Fluorescent Based Chemii stris rss scant 6th aves HERA SRS MS SEARS A 2 Fluorescence Detection and Data Collection 0 0 0 0 ccc cee nes A 4 Mathematical Transformations 0 0 0 eee een e eens A 5 Real Time Data Analysis oo 4accand cone eet ie ete oo ARE ENEE Oak nes A 7 Importing and Exporting Plate Document Data Importing Plate Document Setup Table Files
69. the either the background plate or the sample block module may be contaminated To determine the location of the contamination on the sample block Step Action 1 If not already open open the plate document for the background run 2 From the toolbar click the Hide Show System Raw Data Pane button E The SDS software displays the raw data pane for the background run 3 Select all wells in the plate document Inspect the raw background data for an aberrant spectral peak or peaks Wells producing raw spectra that exceed 2500 FSU are considered irregular and could be contaminated The following figure illustrates the raw data produced by a run on a sample block module containing a contaminated well Amplitude E Contamination 7 Asani 5 Identify the location s of the contaminated well s on the sample block by selecting increasingly smaller regions of the plate document see below 1 2 3 4 5 6 7 8 g 10 11 12 Raw Data Calibration Data Amplitude a The raw data from the selected wells contains the peak o O The contaminated well must be in columns 7 12 m x c ti ce ci ct 5 8 8 B 8 8 8 n 8 8 Troubleshooting To determine the location of the contamination on the sample block continued Step Action Example continued 2
70. the plate document is analyzed Forgot to add a reagent to the plate such as enzyme or master mix Programmed the plate document with the wrong thermal profile a From the Real Time tab of the plate document click Stop b Determine how far into the run the instrument has progressed c Based on the state of the run determine whether the plate can be re run d If necessary eject the plate and add the missing reaction component e If desired re run the plate by re creating the plate document 4 26 Run Setup and Basic Operation After the Run Analyzing the The run can be analyzed immediately following the completion of the run Run Data To analyze data from a plate containing assays for See Page Allelic Discrimination 5 3 Absolute Quantification 6 3 Dissociation Curve Analysis 6 17 Ejecting a Plate The instrument tray must be opened from the plate document that is connected to the Opening Closing 900HT instrument View the instrument status icon to determine the plate document the Instrument Tray tO use to eject the plate Instrument Status Action Fil Connected to PlateName From the Window menu of the SDS software select the plate document connected to the instrument From the plate document select the Instrument tab Click the Plate Read or Real Time tab Click Open Close From the File menu of the SDS software select New Click
71. used in your laboratory These profiles assist users in planning for instrument waste handling and disposal Read the waste profiles and all applicable MSDSs before handling or disposing of waste IMPORTANT Waste profiles are not a substitute for MSDS information As the generator of potentially hazardous waste it is your responsibility to perform the actions listed below Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial or national regulations Note Radioactive or biohazardous materials may require special handling and disposal limitations may apply Obtaining Material Safety Data Sheets MSDS About MSDSs Ordering MSDSs some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are prominently displayed on the labels of all chemicals Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update MSDSs provide you with the safety information you need to store handle transport and dispose of the chemicals safely We strongly recommend that you replace th
72. 1 7900HT Instrument D Comm Power Computer Keyboard Port Hand held Bar Code Reader E Communication Computer Serial Port 2 Plate Handler Port C F Ethernet Network Computer Ethernet Port Ga Comm Power Computer ISA Card 1 Fixed Position Bar Code Reader H Comm Power Bar Code Reader Cable Keyboard not shown a See the figure below Power supply Bar Code Reader Cable Power GR2067 Product Overview 2 11 2 12 Product Overview Section Getting to Know the Software In This Section This section contains the following information Topic See Page Sequence Detection System Software Components and Features 2 14 Managing Sequence Detection System Data 2 15 Working with SDS Software Files 2 16 Product Overview 2 13 Sequence Detection System Software Components and Features Software The ABI PRISM 7900HT Sequence Detection System uses several software Components applications to set up run and analyze experiments completed on the 7900HT 2 14 Product Overview instrument Application Function SDS Software Constructs and edits plate document files sds files Performs initial and end analysis of raw data from allelic discrimination and absolute quantification runs Saves prints and exports run data Automation Controller Software Controls and coordinates the action of the 7900HT instrument and the automation module Initiate
73. 3 Performing a Pure Dye Run 7 17 Adding Custom Dyes to the Pure Dye Set 7 21 Verifying Instrument Performance Using a TaqMan RNase P Plate 7 24 Section Maintaining the Plate Handler 7 27 Adjusting the Sensitivity of the Plate Sensor Switch 7 28 Aligning the Plate Handler 7 32 Aligning the Fixed Position Bar Code Reader 7 40 Cleaning and Replacing Gripper Finger Pads 7 43 Section Maintaining the Computer and SDS Software 7 45 General Computer Maintenance 7 46 Maintaining the SDS software 7 48 System Maintenance 7 1 Recommended Maintenance Schedule Maintenance The following table includes a list of tasks that should be performed on the Schedule ABI PRISM 7900HT Sequence Detection System regularly Interval Task See Page Weekly Archive SDS Files 7 46 Perform a Background Run 7 13 Monthly Check and if necessary replace Gripper Finger Pads 7 43 Defragment the Computer Hard Drive 7 47 Semi Annually Perform a Pure Dye Runa 7 17 Check Applied Biosystems Web Site for Software Updates 7 48 a Perform a background run prior to each Pure Dye Run 7 2 System Maintenance Section Maintaining the 7900HT Instrument In This Section This section contains the following information Topic See Page Replacing the Sample Block 7 4 Changing the 7900HT Plate Adapter 7 9 Decontaminating the Sample Block 7 11 Performing a Background Run 7 13 Performing a Pure Dye Run 7 17 Adding Cus
74. 7900HT Sequence Detection System Although most of the maintenance will be completed by an Applied Biosystems service engineer this section discusses important issues that you should understand System Maintenance 7 45 General Computer Maintenance Maintenance The computer connected to the 7900HT instrument requires regular maintenance to Schedule ensure reliable operation of the ABI PRISM 7900HT Sequence Detection System Archiving SDS Files 7 46 System Maintenance components Applied Biosystems recommends the following tasks as part of routine maintenance of the computer system Maintenance Task Perform Archive or Remove Old SDS Files Weekly Defragmenting the Hard Drive Monthly or before fragmentation reaches 10 Upgrading the Operating System Software When available advisable Upgrading the 7900HT SDS Software When available Developing a Data Management Strategy Applied Biosystems recommends developing a strategy for dealing with the files produced by the SDS software During a single day of real time operation the ABI PRISM 7900HT Sequence Detection System can generate over 200 MB of data Without a strategy for distributing and archiving SDS related files the 7900HT instrument can easily fill the hard drive of the computer within just a few weeks of operation See Managing Sequence Detection System Data on page 2 15 fora discussion of management strategies To conserve space on the computer
75. About Outliers Samples that did not cluster tightly may Contain rare sequence variations Contain sequence duplications Not contain a crucial reagent for amplification the result of a pipetting error Before You Begin Using SDS For specific instructions on any procedure described within this section refer to the Online Help online help accompanying the SDS software To get help at any time during the procedure click a help button located within the dialog box or window in which you are working Examples in The illustrations and screenshots that appear within this chapter were created from a This Section plate containing Pre Developed TaqMan Assays and Reagents for Allelic Discrimination run to screen 6 human genomic DNA samples HD 1 2 3 4 7 8 for 2 targets CYP 2C9 2 and CYP 2C19 2 Each well of the plate contains 1 uL DNA 1X TaqMan Universal PCR Master Mix forward and reverse primers and FAM and VIC labeled TaqMan probes The following figure illustrates the arrangement of the assays unknown samples and no template control NTC wells on the plate o O O fete sous fobetototatetetetote GR2107 Dy PDAR CYP 2C9 2 PDAR CYP 2C19 2 Note The probes used in the example experiment were designed using the Primer Express Primer Design Software and by following the guidelines explained in Assay Development Guidelines on page C 2 IMPORTANT The SDS software does not require that a
76. Applied Biosystems Group Applied Biosystems warrants to the customer that for a period ending on the earlier of one year from the completion of installation or fifteen 15 months from the date of shipment to the customer the Warranty Period the ABI PRISM 7900 HT Sequence Detection System purchased by the customer the Instrument will be free from defects in material and workmanship and will perform in accordance with the installation specifications set forth in the system specifications sheet which accompanies the instrument or which is otherwise available from an Applied Biosystems sales representative During the Warranty Period if the Instrument s hardware becomes damaged or contaminated or if the Instrument otherwise fails to meet the Specifications Applied Biosystems will repair or replace the Instrument so that it meets the Specifications at Applied Biosystems expense However if the thermal cycling module becomes damaged or contaminated or if the chemical performance of the Instrument otherwise deteriorates due to solvents and or reagents other than those supplied or expressly recommended by Applied Biosystems Applied Biosystems will return the Instrument to Specification at the customer s request and at the customer s expense After this service is performed coverage of the parts repaired or replaced will be restored thereafter for the remainder of the original Warranty Period This Warranty does not exten
77. Click the Description text field and type a brief 32 character description of the assay 5 Click the Reporter Dye and Quencher Dye drop down lists and select the appropriate dyes for the assay Note If creating a detector fora SYBR Green 1 assay set the Quencher Dye drop down list to Non Fluorescent Note If using a custom dye not manufactured by Applied Biosystems you must create and run a pure dye plate for the custom dye before it can be applied to a detector see Adding Custom Dyes to the Pure Dye Set on page 7 21 6 Click the Color box select a color to represent the detector using the Color Picker dialog box and click OK 7 Optional Click the Notes text field and type any additional comments for the detector up to 200 characters x xij N Name text field Name CYP 2C9 2 Allele 1 Name RNase P Description Pre Developed Taqman Assay Reage Description Human RNase P Gene Taghlan Assay Description text field Reporter FAM Reporter FAM x Dye drop down Quencher Non Fluorescent Quencher TAMRA lists Color E Color E Color box Notes This is an example of a detector Notes This is an example of a detector constructed for an allelic constructed for an absolute d discrimination assay quantification assay Notes text field Created Jun 19 2001 7 54 41 PM Created Jun 13 2001 8 44 41 PM i Last Modified Jun 19 2001 7 54 41 PM Last Modified
78. Contacting Technical Support for more information Run your experiment Note If conducting a quantitative PCR experiment consider the use of replicate assays to enhance the precision of you data C 4 Designing TaqMan Assays Design Tips for Allelic Discrimination Assays Discrimination by By using different reporter dyes cleavage of multiple probes can be detected in a Multiple Probes single PCR One application of this multi probe capability is to use allele specific probes to distinguish genetic polymorphisms Bloch 1991 Lee et a 1993 Probes that differ by as little as a single nucleotide will exhibit allele specific cleavage This is true even for probes with a reporter on the 5 end and the non fluorescent quencher on the 3 end Bloch 1991 TaqMan Probe IMPORTANT When designing probes it is important to consider probes from both strands Design Guidelines Follow the guidelines in the table below for designing TaqMan MGB probes Priority Guideline 1 Avoid probes with a guanine residue at the 5 end of the probe A guanine residue adjacent to the reporter dye will quench the reporter fluorescence even after cleavage Select probes with a Primer Express software estimated Tm of 65 67 C Make the TaqMan MGB probes as short as possible but no fewer than 13 nucleotides in length Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more
79. DHOST window closes 10 Replace the cover for the fixed position bar code reader from step 1 on page 7 40 Cleaning and Replacing Gripper Finger Pads When to Perform The adhesive used to affix bar code labels to certain brands of microplates can build up on the gripper pads of the Zymark Twister Microplate Handler Over time the residue can cause the gripper pads to stick to the microplates while handling them causing misfeeds To prevent buildup inspect the gripper pads monthly and clean or replace the pads as needed Materials Required The following materials are required to replace the finger pads Material Part Number Finger Pad Replacement Kit containing 10 finger pads 4315472 Flat blade screwdriver small Phillips head screwdriver small lsopropanol in a squeeze bottle CHEMICAL HAZARD Isopropanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin and cause irritation It may cause central nervous system effects such as drowsiness dizziness and headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Cleaning the To clean the finger pads wipe each pad thoroughly with Isopropanol until the residue Finger Pads has been resolved If the pads appear rough or the adhesive cannot be removed replace the pads as describ
80. DS Plate Document Files Product Overview 2 7 Bar Code Readers Description The ABI PRISM 7900HT Sequence Detection System incudes two bar code readers Locations of the Bar Code Readers for data entry and plate recognition ahand held bar code reader for scanning plates manually a fixed position bar code reader for automated scanning of plates as they are loaded into the instrument available with automation accessory only Both bar code readers use a 488 nM laser to scan plates and are capable of reading Code 128 alpha numeric which supports 128 ASCII characters The following figure illustrates the locations of the bar code readers in the system Hand Held Bar Code Reader To computer Splitter keyboard port l To keyboard GR2015 Using the PAULIE LASER HAZARD Exposure to direct or reflected laser light can burn the retina Bar Code Readers 21d leave permanent blind spots Never look into the laser beam Remove jewelry and anything else that can reflect the beam into your eyes Protect others from exposure to the beam 2 8 Product Overview For directions on See Page Using the Hand Held Bar Code Reader 3 19 Aligning the Fixed Position Bar Code Reader 7 40 Zymark Twister Microplate Handler Description The Zymark Twister Micro
81. Detection System is sequential 7900HT Instrument each component must be activated in a specific order for the system to initialize properly If performed out of sequence the components may not be able to establish the necessary communication connections S2 Applied BS Bey Sems Status lights 7900HT instrument power button Monitor power button api PRISM Zymark Twister Microplate Computer Handler power button in the rear power button IMPORTANT Turn on the power to the instrument and the plate handler at least 10 min before use When activated the instrument heats the sample block cover to 105 C If a run is started before the heated cover reaches 105 C the instrument will pause until it reaches the optimal temperature before commencing the run To activate the components of the ABI PRISM 7900HT Sequence Detection System Step Action 1 Turn on the monitor and computer 2 Turn on the Zymark Twister Microplate Handler by pressing the power switch located on the back panel of the plate handler see below I N OLAU Ee PHYSICAL HAZARD Keep clear of the arm when activating the plate handler Once activated the arm automatically moves to its home position l TI Power switch 3 O oeeo esz ome
82. F4 F5 F6 F7 Fs F9 F10 F11 F12 2 E a S m m S E 2 m 2 E ae T E a x a x T 6 x o ai an se ee 5 6 7 8 9 10 11 12 Raw Data Calibration Data E E E E E E 8 E A mplitude A2 AB A4 A5 AB A7 AB Ag A E E E E E E E E E B fo B2 B3 B4 BS BB B7 BS Bg B10 B11 B12 Jia E E E E E E E E E E fc c2 c3 c4 c5 c6 c7 cs co c10 cn c12 E E E E EE E E E E E D fo D2 D3 D4 D5 D6 D7 DS D9 D10 D11 D12 D 30 E E E E EE E E E E E Wavelength E fe4 E2 E3 E4 E5 E6 E7 ES E9 E10 E11 E12 Temperature C 60 E E E E E E E E E 40 Ffa F2 F3 F4 F5 F6 F7 FS F9 F10 F11 F12 40 E E E E EE ti E E E 20 G Io1 62 63 64 65 66 67 68 69 eo fen 612 E E E H Jus H2 H3 H4 HS HG H7 HB HO H10 H11 H12 Time s an Time s nn Time s ann Time s an The raw data from The raw data from The raw data from By selecting each the selected wells does not contain the peak The contaminated well must be in the last four wells of columns 7 12 the selected wells contains the peak The contaminated well must be in the last four wells of columns 10 12 the selected wells contains the peak The contaminated well must be in the last two wells of columns 10 12 of the wells from the last two wells of columns 10 12 the location of the contaminated well G10 is determined Repeat step 4 until you identify the loc
83. Gi o G Mra Mra Me Me Me Mei Me p ERE EERE HEEE EEEE EEEE PB ee EE Sizing mM ri M ri M nR M nr Mnr Mnr MrR M nR MRM AR eBRA eR AR A nr M nr Mnr M nri M nR M nr M nr M nr M nR 4 button Position Sample Name Detector Task Ct Quantity Qty mean __ Oty stao A UNKNSK RNaseP Unknown 2 Click and drag the grey divider at the bottom of the plate document to restore the table pane to its original size using the action described in Exercise 1 above 3 Using the sizing buttons maximize minimize other elements of the plate document until comfortable using the feature Getting Started 3 15 Lesson 3 Using the Plate Grid 3 16 Getting Started Exercise 3 Hiding and Showing Panes Views and Plots You can also toggle the presence of the plate document panes views and plots using the icons in the Display toolbar To illustrate this feature hide and show the table pane as follows Step Action 1 From the Display toolbar click the Show Hide Table Pane button H The software removes the table pane from the plate document 2 Click the Show Hide Table Pane button Hf again to show the table pane The software restores the table pane to the plate document The display toolbar can be particularly useful for manipulating information shown in the plate document See Using the Display Toolbar on page 3 9 for a list of the other icons of the display toolbar and the panes they control 3 Pr
84. However if creating a plate document to run a single plate scan or type the bar code for the plate into the Barcode text field 4 Click OK The software displays a new plate document with the appropriate attributes 5 Create and copy detectors and markers to the new plate document as described on page 4 7 4 6 Run Setup and Basic Operation Step 2 Applying Detectors and Markers to the Plate Document Creating Detectors Before a plate document can be used to run a plate it must be configured with detector information for the experiment and marker information if performing allelic discrimination A detector is a virtual representation of a TaqMan probe in a master mix used for detection of a single target nucleic acid sequence or the SYBR Green Double Stranded DNA Binding Dye 1 used for the detection of double stranded DNA Before using the plate document you must create and apply detectors for all assays present on the plate To create detectors for the plate document Step Action 1 From the Tools menu of the SDS software select Detector Manager The Detector Manager dialog box appears 2 Click File and from the drop down list select New The Add Detector dialog box appears 3 Click the Name text field and type a name for the detector IMPORTANT The name of the detector must be unique and should reflect the target locus of the assay such as GAPDH or RNase P 4 Optional
85. Instrument Progress Stopping the Sequence Detector from the Automation Controller Software EKjecting a Plate Opening Closing the Instrument Tray After the Run Analyzing the Run Data Once all plate documents have been loaded into the Plate Queue and the Instrument Control options are configured you may start the plate queue To begin the plate queue click Start Queue from the Plate Queue tabbed page The ABI PRISM 7900HT Sequence Detection System loads the first plate scans the bar code and the begins the run Note Before starting a real time run the instrument may pause up to 15 minutes to heat the heated cover to the appropriate temperature The Automation Controller Software displays the progress of the current run in the Thermal Status tab See page 4 25 for an explanation of the Thermal Status tab To stop the plate queue click the Stop button on the Automation Controller Software dialog at any time If the Stop button is clicked Then the instrument while the plate handler is handling a plate aborts the run and moves the plate handler to the home position after a plate has been loaded into the instrument but before the run has started aborts the current run ejects the plate and moves the plate handler to the home position after the 7900HT instrument has started a run aborts the current run ejects the plate and moves the plate handler to the home position IMPORTANT S
86. Jun 13 2001 8 44 41 PM Creation and Modification Maker CYP 2C09 2 Marker date stamps TA Allelic Discrimination Detector Absolute Quantification Detector Run Setup and Basic Operation 4 7 Copying and Applying Detectors to the Plate Document To create detectors for the plate document continued Step Action 8 Click OK to save the detector and return to the Detector Manager dialog box The software saves the new detector and displays it in the detector list 9 Repeat steps 2 to 8 to create detectors for all remaining assays on the plate Note Click the 3 button for information on the features of the Detector Manager dialog box or to view the procedures for editing deleting or searching for detectors 10 Choose from the following If constructing a plate document for Then absolute quantification copy the detector s to the plate document as explained in the procedure below allelic discrimination create markers to the plate document as explained on page 4 9 IMPORTANT Once copied to the plate document a detector is no longer linked to the corresponding entry in the Detector Manager Therefore if a detector is modified in the Detector Manager after it has been copied to a plate document the detector must be removed from the plate and copied again to update the plate document with the changes To copy and assign the detectors to the plate document
87. N JO OO OO OO OO QUOD OUQO00 O O O O O O O O O O O00 CO COCOC00000 OO0OO00000000000 ele oO OO CO OO OO O aE OO OO CO OO OO OO OO GR2107 6 6 Real Time Analysis Analysis Checklist Where You Are in The following checklist illustrates your current position in the overall procedure the Procedure Done Step Procedure See Page J 1 Create an absolute quantification plate document 4 6 J 2a a Create detectors for the absolute quantification probes 4 7 b Copy the detectors to the plate document 4 8 S 3a a Configure the plate document with detector tasks 4 11 NTC Standard and Unknown b Assign quantities to the wells of the plate document that 4 12 contain standards J 4 a Program the method for the absolute quantification run 4 13 b If performing an assay in which you would like to collect 4 16 dissociation data add a temperature ramp to the thermal profile to perform a dissociation curve analysis J 5 Choose from the following If running Then a single plate continue to step 7 the first plate in a series Save the plate document as an ABI PRISM of plates with identical SDS Template Document as explained on assay configurations page 4 17 J 6 Create a plate document from the template created in 4 18 step 5 J 7 Configure the document with sample names
88. Nase P No Template RNase P RNase P ISTND 2500 __ RNase P STND 5000 RNase P RNase P RNase P RNase P RNase P RNase P _ RNase P RNase P RNase P RNase P RNase P ane i acces Mo Mhie M Table elements Unknown HEETE 4641 368 Paos er 478 2128 _ Unknown 24 617865 4774 804 478 2128 Unknown 24 523537 5091 7935 478 2128 Unknown 24 61123 4796 439 478 2128 Unknown 24 498795 5178 3677 4905 1187 478 2128 Unknown 24 54585 5014 958 4905 1187 478 2128 Unknown 24 649157 4674 0693 Unknown 24600046 4833 135 4905 1187 478 2128 Unknown 24 541927 5028 384 4905 1187 478 2128 NTC 40 0 0 0 Standard 26 59769 1250 0 Standard 25 530634 2500 0 Standard 24 657196 5000 0 Standard 23 558832 10000 0 Standard 22 502737 200000 l Unknown 23 531593 10009 766 9800 406 493 38834 Unknown 23 536495 9976 389 493 38834 Unknown 23 616756 9445 412 Unknown 23 545746 9913 696 Unknown 23 681985 9671 884 Unknown 23 623846 9399 892 4 Unknown 23 554886 9852 144 Unknown 23 628742 9368 584 _ AA DADIN OeaTwv ara AAND AS INA ANNA a Column Displays Position The coordinate position of the well on the plate Sample Name The sample name applied to the well Note See page 4 19 for information on applying sample names to the plate document Detector The name of the detector assigned to the well Note See
89. OK Click the Instrument tab Click the Plate Read or Real Time tab Click Open Close zi Disconnected P9QAaqonwml QqQans5 Run Setup and Basic Operation 4 27 Disconnecting the The SDS software has the ability to halt all communications with the 7900HT Software from the instrument The disconnect option is designed to permit the simultaneous operation Instrument Of both the SDS software and the Automation Controller Software Because both programs control the ABI PRISM 7900HT Sequence Detection System one program must relinquish control of the 7900HT instrument before the other can be used to operate it To Then disconnect the software from the instrument a From the SDS software click the Instrument tab of the open plate document b Click the Real Time or Plate Read tab c Click Disconnect Note Once disconnected the software neither monitors nor controls the 7900HT instrument reconnect the software once disconnected to reconnect to an open plate document a From the File menu select Close to close the plate document b From the File menu select Open c From the Look In text field navigate to and select the plate document of interest d Click Open Upon opening the plate document the software re establishes the 7900HT instrument connection to reconnect to an new plate document select New from the File menu Upon creation of the plat
90. Open Close The instrument tray rotates to the OUT position f From the File menu select Exit The SDS software exits 2 Remove the four screws attaching the plate holder to the plate arm Unscrew Unscrew 3 Remove the plate adapter from the instrument tray Note If changing sample block formats for example replacing a 384 well sample block with a 96 well version store the plate adapter with the sample block module of the same format System Maintenance 7 9 7 10 System Maintenance To replace the 7900HT instrument plate adapter continued Step Action 4 Place the new plate adapter into the instrument tray with the A1 label in the rear left corner see below IMPORTANT Make sure to install the correct version of the plate adapter 384 or 96 well for the plate format you intend to use The plate adapters are labeled for the consumable format they support Well A1 Label 384 or 96 well 2 2 Replace and tighten the four screws in the order shown below IMPORTANT The order in which the screws are tightened is important to ensure proper alignment of the plate to the sample block within the 7900HT instrument Decontaminating the Sample Block
91. Operation Mathematical Transformations Overview Multicomponenting The SDS software performs a series of mathematical transformations on the raw data during an analysis of all end point and real time runs The term raw data refers to the spectral data between 500 nm to 660 nm collected by the SDS software during the sequence detection run The following section describes the fundamental analysis of raw run data performed on both real time and end point run by the SDS software Multicomponenting is the term used for distinguishing the contribution each individual dye and background component makes to the fluorescent spectra detected by the 7900HT instrument During the multicomponent transformation the SDS software employs several algorithms to separate the composite spectra from the raw spectrum and then to determine the contribution of each dye in the raw data First the algorithm eliminates the contribution of background fluorescence in the raw data by subtracting the background component stored within the background calibration file See page 7 13 Next the software employs the extracted pure dye standards see page 7 17 to express the composite spectrum in terms of the pure dye components The figure below shows one composite spectrum that represents a fluorescent reading from a single well that contains the passive reference and two fluorogenic probes labeled with the FAM and VIC reporter dyes and a non fluorescent quencher The
92. RISM Optical Caps flat cap strips only IMPORTANT Do not use MicroAmp Optical Caps or MicroAmp Optical Tubes with the 7900HT instrument The instrument is not designed to run MicroAmp consumables which may damage its internal components if used Note ABI PRISM Optical Reaction Plates are designed specifically for fluorescence based PCR chemistries and are frosted to minimize external fluorescent contamination Before running prepared ABI PRISM Optical Reaction Plates on the 7900HT instrument each plate must be sealed with an ABI PRISM Optical Adhesive Cover Applied Biosystems optical adhesive covers are specifically designed to permit the transmission of light to and from the wells of the optical plate Part No Description Quantity ABI PRism Optical Adhesive Covers 4313663 ABI PRISM Optical Adhesive Cover Starter Kit 20 Covers Includes 20 ABI PRISM Optical Adhesive Covers an Applicator and a ABI PRISM Optical Cover Compression Pad 4311971 ABI PRism Optical Adhesive Covers 100 Covers 4323032 ABI PRismM Optical Caps 8 Caps Strip 300 Strips Pkg 2400 Caps Pkg 384 Well Optical Reaction Plates 4309849 ABI PRISM 384 Well Clear Optical Reaction Plate with 50 Plates Barcode code 128 4326270 10 Pack ABI PRISM 384 Well Clear Optical Reaction Plate 500 Plates with Barcode code 128 Includes 10 ABI PRISM 384 Well Clear Optical Reaction Plate with Bar
93. Running an Individual Plate 4 21 Saving the Plate Document 4 22 Preparing and Running a Single Plate 4 23 Operating the 7900HT Instrument Using the SDS Software 4 25 After the Run 4 27 Section Running Multiple Plates Using the Automation Controller 4 29 Adding Plate Documents to the Plate Queue for Automated Operation 4 30 Adding a Plate Document to the Plate Queue from the SDS Software 4 31 Creating Plate Documents Using the Template Batch Utility 4 32 Running Plates Using the Automation Controller Software 4 34 Loading Plates onto the Automation Module 4 36 Operating the 7900HT Instrument Using the Automation Controller Software 4 38 After the Run 4 38 Run Setup and Basic Operation 4 1 Before You Begin Background Chapters 5 and 6 include brief discussions of the experiments that can be performed Information using the 7900HT instrument Before beginning you may want to review the appropriate chapter for your experiment Run Type Experiment See Page End point Allelic Discrimination 5 3 Real time Absolute Quantification 6 3 Dissociation Curve Analysis 6 17 Getting More The SDS software features an online help system that can guide you through the Information from procedures for setting up performing and analyzing runs To get help at any time the Online Help click the 3 button located within the dialog box or window in which you are working Maximizing For end point applications such as allelic
94. System 5 End Point Analysis Describes how to analyze data from allelic discrimination experiments 6 Real Time Analysis Describes how to analyze data from absolute quantification and dissociation curve experiments 7 System Maintenance Explains how to perform both routine and incidental system maintenance for the components of the ABI PRISM 7900HT Sequence Detection System 8 Troubleshooting Contains tips for troubleshooting problems with the ABI PRISM 7900HT Sequence Detection System 9 User Bulletins This chapter is reserved for user bulletins A Theory of Operation Describes the principles behind the operation of the ABI PRISM 7900HT Sequence Detection System B Importing and Exporting Explains the Import Export function of the SDS software Plate Document Data and also diagrams the structure and annotation of setup table files C Designing TaqMan Assays Contains brief instructions for designing TaqMan probe and Sequence Detection primer sets D Kits Reagents and Contains a list of Applied Biosystems kits and Consumables consumables for use with the 7900HT instrument References Contains a bibliography of references for this manual F Contacting Technical Support How to contact Applied Biosystems Technical Support Limited Warranty Statement The Applied Biosystems limited warranty statement Turning on the ABI PRISM 7900HT Sequence Detection System Turning On the The activation of the ABI PRISM 7900HT Sequence
95. System Maintenance To position the fixed position bar code reader continued Step Action 5 Loosen the black positional adjustment knob on the fixed position bar code reader and position the scan head of the reader as far as possible from the plate while maintaining the orientation towards the bar code on the plate see below Scan head of the fixed position bar code reader Black positional adjustment knob 6 While watching the Terminal dialog box slowly adjust the orientation of the fixed position bar code reader until the percent successful reading displays the highest number possible EN Terminal Percent successful reads Note It may be helpful to briefly place a sheet of white paper in front of the plate bar code to view the area scanned by the laser 7 When satisfied with the alignment tighten the black positional adjustment knob on the fixed position bar code reader 8 Restore the fixed position bar code reader to normal operation a From the Edit Configuration dialog box change from Test back to Serial on Line ajalaje 2 HE CMe Test a lt 2 moai Mode drop down list Operatina mode options b From the Device Control dialog box confirm that EEPROM is still selected and click Send c From the New Decision dialog box click YES to save to EEPROM The bar code reader stops scanning the plate bar code and resumes normal operation 9 Click Exit to quit the LDHOST window The L
96. Text appearing between brackets is required information in setup table files All information within the brackets must be present in the setup table file for the SDS software to import it required text Text appearing between braces is optional in setup table files lt tab gt The tab character the equivalent of pressing the Tab key lt cr gt The carriage return character the equivalent of pressing the Enter key IMPORTANT To guarantee a successful importation of the setup table file into a plate document the file can must contain all of the sections in the following table in the order that they appear in this document Setup Table Elements Number Contents Description 1 File Version This line defines the version of SDS Assay Plate File format used to generate the document Format SDS Setup File Version lt tab gt version number lt cr gt Example see SOS OCTU Fale Version 2 2 Plate Size This line defines the number of wells in the plate modeled by the file 384 or 96 Format Output Plate Size lt tab gt number of wells lt cr gt Example wee OCU PE have SaaS 384 3 Plate ID This line defines the ID of the Assay Plate Normally this will be a bar code that is printed on the plate Format Output Plate ID lt tab gt plate id lt cr gt Example Output Plate ID 384N75822034 Importing and Exporting Plate Do
97. UNEASE UNEASE L m E m m E l 2 Click the Zoom Grid button again to restore the plate grid to the original size Practice zooming portions of the plate grid until comfortable using the feature Exercise 4 Resizing Wells Using the Border Lines You can also adjust the size of the plate grid wells by moving the lines in the row or column headers To illustrate this feature Step Action 1 Move the mouse cursor over a border line in the row or column header The mouse cursor becomes a double arrow lt gt 2 Click and drag the mouse cursor to adjust the well to a new width The software resizes all wells in the plate grid to match the new width Mouse cursor P RNaser P RNazeP RNase P RNase P RNazer UNEASE UNEASE UNENSEK UNEASE UNEASE g m E E fs i E I 3 Practice resizing the wells of the plate grid until comfortable using the feature Lesson 4 Using The hand held bar code reader functions as an extension of the keyboard and can be the Hand Held used to automatically type bar codes into the SDS software When the reader is used Bar Code Reader Successfully to scan a bar code it automatically transmits the alpha numeric equivalent of the bar code to the software The software types the bar code text wherever the cursor is active transmits a carriage return the equivalent of pressing the Enter key Exercise Entering Bar Code Information Using the Hand He
98. When to Perform The following procedure describes how to decontaminate the wells of a sample block module The procedure will eliminate residual PCR related products including fluorescent labeled TaqMan probes Clean the sample block as often as needed IMPORTANT If preforming a cleaning or decontamination method other than the one in this manual check with Applied Biosystems first to ensure that the method will not damage the sample block module or the 7900HT instrument Materials Required The cleaning procedure requires the following materials Material Part Number Cotton swabs 10 Sodium hypochlorite bleach solution Isopropanol 100 percent pure 5 32 in Hex key 7 Ne Nai e CHEMICAL HAZARD Sodium hypochlorite bleach is a liquid disinfectant that can be corrosive to the skin and can cause skin depigmentation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CHEMICAL HAZARD Isopropanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin and cause irritation It may cause central nervous system effects such as drowsiness dizziness and headache etc Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves System Maintenance 7 11 Cleaning the Sample To clean the sample b
99. Z zooming allelic discrimination plot plate grid wells 3 18 Zymark Twister Microplate Handler See plate handler Zymark Twister software about 2 14 aligning the plate handler closing 7 39 launching 7 32 testing the plate sensor switch 7 30 5 11 7 32 to 7 39 Index 7 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained Support and applications personnel reaches into 150 countries on six continents For sales office locations and technical support please call our local office or refer to our web site at www appliedbiosystems com or to the Technical Support and Training appendix in this document www appliedbiosystems com Applied Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 12 2001 Part Number 4317596 Rev A4 an Applera business ABI PRISM 7900HT Sequence Detection System Applied Biosystems User Guide
100. ack the plates as explained in page 4 37 Bar code reader is mis aligned Bar code reader is broken Re align the fixed position bar code reader as explained in Aligning the Fixed Position Bar Code Reader on page 7 40 Troubleshooting 8 17 User Bulletins About This Chapter A user bulletin is an advisory issued by Applied Biosystems User bulletins contain new information advances or procedures that may immediately influence your use of Applied Biosystems instruments This section of the user guide is intended as a storage space for any user bulletins you may receive regarding your ABI PRISM 7900HT Sequence Detection System User Bulletins 9 1 Theory of Operation In This Appendix This appendix discusses the following topics Topic See Page Fluorescent Based Chemistries A 2 Fluorescence Detection and Data Collection A 4 Mathematical Transformations A 5 Real Time Data Analysis A 7 Theory of Operation A 1 Fluorescent Based Chemistries Fundamentals of the The PCR reaction exploits the 5 nuclease activity of AmpliTaq Gold DNA 5 Nuclease Assay Polymerase to cleave a TaqMan probe during PCR The TaqMan probe contains a A 2 Theory of Operation reporter dye at the 5 end of the probe and a quencher dye at the 3 end of the probe During the reaction cleavage of the probe separates the reporter dye and the quencher dye which results in increased fluorescen
101. actice hiding and showing the other plate document elements by clicking other buttons in the Display toolbar until comfortable using the feature The plate grid see below is an important interface tool for the SDS software The software uses the grid to convey information about the plate and allows you to select specific wells for viewing and analysis The following exercises will teach you how to use and modify the elements of the plate grid mi f oo aa o ay x m gt w T Sy on m fez as per m o wo M N S pos le Plate grid Ei A B fe D E F G H l J K L M N o P yb a a yet ped eh Jed e w D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D a 7a oa 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 iL Jel Jel fel fel Jet Jel Jet fel Jet jel Jet fel Jel Jel Jel ies 2 23 2 2 2 2 2 2 2 2 2 2 2 2 A Q 2 iL Sel Jel fel fel fet Jel Jet fel Jet Jel Jet Jel Jel Jel Jel A 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 iL Jel eb seh fet Jeb deh deh Jel Jel jel Jel fel fel fel Jel IR 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 P yeh deh fel deh feb seh fed deh Jeb fel eh deh Jel fel el 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Jel Jel fel_fellelleLleLJeL_teL_felJelJeL_JeLJeL Jel 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Leh seh feb det deh seh eh deh Jeb fel Jet deh Jel fet JeL D D D D D D D D D D D D D D D D LJeLJeLJelLseL_leL_JeLieL_leLieL_feljeljeL_jeLJeL Jel Im D D D D D 2 2 2 2 2 2 2 2 2 2 D eFC CRR
102. age From the Real Time or Plate Read tab click Open Close The instrument tray rotates to the OUT position Place a plate into the instrument tray as shown below Before loading the plate onto the instrument tray make sure that The associated plate document is open in the SDS software The plate has been sealed using an optical adhesive cover HAT Et PECYOO1DL3 j Well A1 GR2111 IMPORTANT The A1 position is located in the top left side of the instrument Do one of the following If performing a real time run click Start If performing a end point run click Plate Read The instrument tray rotates to the IN position and the instrument performs the run or plate read Note Before starting the run the instrument may pause up to 15 minutes to heat the heated cover to the appropriate temperature Note For more information on the elements of the Real Time and Plate Read tabs click the button and see the SDS software online help The following options are available during and after the completion of the run To See Page Monitor the progress of the run 4 25 Stop the run 4 26 IMPORTANT _ If you must stop a run in progress for any reason carefully read the instructions on page 4 26 before halting the run Open the instrument tray after the run 4 27 Analyze the run data after the run is
103. al minutes The specific binding characteristic of the SYBR Green 1 Dye permits the 7900HT instrument to monitor the hybridization activity of the nucleic acids present in the sample During the run the instrument records the decrease in SYBR Green fluorescence resulting from the dissociation of dsDNA After the run the SDS software processes the raw fluorescence data from the SYBR Green 1 Dye to generate a more meaningful representation of the relationship between spectral change and temperature for the dissociation curve run Multicomponenting and Normalization The first mathematical transformation involves the conversion of the raw data expressed in terms of Fluorescent Signal vs Wavelength using the extracted pure dye standards to pure dye components After the dye components have been identified the software determines the contribution of each dye in the normalized data using the multicomponent algorithm see Multicomponenting on page A 5 for a complete description of the process Finally the software normalizes the data using the component of the passive reference dye as shown below R R SYBR R PassiveReference Derivation of Dissociation Curve Data The SDS software then computes the first derivative of the normalized data R for each reading taken by the 7900HT instrument during the temperature ramp The resulting derivative data R is the rate of change in fluorescence as a function of temperature see b
104. alysis 6 15 6 16 Real Time Analysis Section Dissociation Curve Analysis In This Section This section contains the following information Topic See Page Overview 6 18 Before You Begin 6 19 Analysis Checklist 6 20 Analyzing the Run Data 6 21 Determining Tm Values for the Analyzed Run 6 22 After the Analysis 6 24 Real Time Analysis 6 17 Overview About Dissociation Curve Analysis Employing the SYBR Green 1 Dye Mathematical Transformations 6 18 Real Time Analysis The ABI PRISM 7900HT Sequence Detection System supports dissociation curve analysis of nucleic acids using SYBR Green 1 double stranded DNA binding dye chemistry The objective of dissociation curve analysis is to accurately determine the melting temperature T of a single target nucleic acid sequence within an unknown PCR sample Typical uses of dissociation curves include detection of non specific products and primer concentration optimization Dissociation curve analysis on the 7900HT instrument is made possible through the use of the fluorogenic SYBR Green 1 double stranded DNA binding dye chemistry see page A 3 Dissociation curves are commonly performed following the PCR stage of a SYBR Green experiment to screen for non specific products To generate the data needed to create a curve the 7900HT instrument performs a programmed temperature ramp in which it slowly elevates the temperature of the plate over sever
105. and detection Components Of fluorescent chemistries see page 2 2 The figure below illustrates the major Automated Plate Handling System Heated Clamp 2 4 Product Overview subcomponents of the instrument described in further detail below Laser CCD Optics camera System Reader Heated clamp Tray Automated plate Door handling system Sample block module Do not remove the cover to the ABI PRISM 7900HT Sequence Detection System Only a qualified Applied Biosystems service engineer may repair or adjust the internal components of the 7900HT instrument Failure to comply can result in serious injury and or damage to the instrument ALGOLIN PHYSICAL HAZARD Keep hands and loose clothing away from the instrument tray and door at all times during instrument operation The 7900HT instrument contains several internal components that can cause serious physical injury The 7900HT instrument features an automated plate handling system to provide easy loading and removal of plates from the instrument In combination with the automation module the plate handling system allows unattended operation of the instrument AANE PHYSICAL INJURY HAZARD Hot Surface Use care when working around this area to avoid being burned by hot components The 7900HT instrument features a heated clamp that provides optimal heat transfer and uniform heating during thermal cycling When the instrument tray loads a
106. and most plots Allelic Discrimination Raw Data and Background plots To configure the display settings for the plate document Step Action 1 From the View menu select Display Settings 2 From the Display Settings dialog box click the help button for further instructions on modifying the display settings The software allows you to save any changes to the appearance of the plate document however it does not save the calls made during the analysis To save the plate document select Save from the File menu The SDS software can print a report of the analyzed data containing individual or multiple elements of the plate document To print a report of the plate document data Step Action 1 From the File menu select Print Report 2 From the Print Report dialog box click the help button _ for instructions on setting up previewing and printing the report Exporting Plate Document Data as a Tab Delimited Text File The SDS software can export raw or analyzed data in tab delimited txt format for all or a select group of wells on a plate document The exported files are compatible with most spreadsheet applications and programs that can read tab delimited text To export run data as a tab delimited text file choose one of the following for further instructions See Exporting Plate Document Data on page B 9 Click the help button 7 within the table view
107. andle multiple documents simultaneously however the processing speed of your computer will decrease with each open document For that reason Applied Biosystems recommends limiting the number of open documents to 10 3 Configure the New Document dialog box with the following settings Drop Down List Select Assay Absolute Quantification Container 384 Wells Clear Plate Template Blank Template 4 Click OK The software displays a new plate document with appropriate attributes Exercise 2 Saving a Plate Document The Save command stores any changes to the plate document setup information and display settings The following procedure explains how to save the open plate document To save the plate document Step Action 1 Choose one of the following options Click the Save button l from the General toolbar or From the File menu select Save As From the File of type drop down list select ABI PRism SDS Single Plate sds Click the File name text field and type Practice Click Save The software saves the plate document to a file entitled Practice sds Note Do not close the plate document at this time Exercise 3 Opening a Plate Document In this exercise you will be opening a template file that you will use in the following exercises To open a plate document Step Action 1 Choose one of the following options Click the Open button from the General toolba
108. are required to perform the RNase P run Material Part Number TaqMan RNase P Instrument Verification Plate See page D 4 Centrifuge with plate adaptor Preparing a RNase P To prepare a plate document for the RNase P plate Plate Document Step Action 1 Remove the TaqMan RNase P Instrument Verification Plate from the freezer and allow it to thaw to room temperature Launch the SDS software 3 From the File menu select New The New Document dialog appears 4 Configure the New Document dialog box as follows Drop Down List Select Assay Absolute Quantification Container lt the appropriate plate format gt Template lt the template file of the appropriate plate format gt 5 If desired enter the bar code information into the plate document as follows a Click the Barcode text field b Remove the RNase P plate from the packaging and scan its bar code using the hand held bar code reader 6 Click OK The software creates a plate document Note Do not modify the RNase P plate document The template is pre programmed with detector and method information for the run 7 Save the plate document as follows a From the File menu select Save The Save dialog appears b Click the Barcode text field and either Type aname or bar code number for the plate and click Save Using the hand held bar code reader scan the bar code number c From the Files of type d
109. as explained on page 6 12 Real Time Analysis 6 11 Eliminating Outliers For any PCR experimental error may cause some wells to amplify insufficiently or not 6 12 Real Time Analysis at all These wells typically produce C values that differ significantly from the average for the associated replicate wells If included in the absolute quantification calculations these outliers can potentially result in erroneous measurements Visualizing Outliers To ensure precise absolute quantification replicate groups must be carefully scrutinized for outlying wells The C vs Well Position view of the Amplification Plot allows you to examine each set of replicate wells for outliers To visualize the replicate groups for outlying amplification Step Action 1 From the Plot drop down list select Ct vs Well Position The SDS software displays the results data in a Well versus Threshold Cycle plot 2 Verify the uniformity of each replicate population by comparing the groupings of C values for the wells that comprise the set Are outliers present Then Yes a Determine and record the well numbers of all outlying wells b Go to the next step No go to the next step 3 Check for remaining detectors present on the plate for outliers a Click the Detector drop down list and select another detector b Repeat step 2 until each detector has been checked for outliers 4 If outliers are prese
110. as it is saved in tab delimited format and is configured according to the file structure explained on page B 4 To export a blank setup table file using the SDS software Step Action 1 Launch the SDS software 2 From the File menu select New 3 Configure the New Document dialog box with the correct assay type and plate format for your experiment and click OK 4 From the File menu select Export 5 From the Look In text field of the Export dialog box navigate to the directory you would like to receive the exported file From the Export drop down list select Setup Table Select the All Wells radio button Click the File name text box and type a name for the file O OINI O Click Export The software exports the setup table data for the empty plate document as a tab delimited text file 10 Configure the setup table file with plate document information detector task marker and sample data as explained on page B 3 B 2 Importing and Exporting Plate Document Data Configuring the Setup Table File with Plate Document Information The second step in the procedure is to import the setup table file into a secondary application configure it with sample and detector information and then save the completed setup table file in tab delimited format To configure the setup table file with information Step Action 1 Launch the application that you want to use to edit
111. at can reflect the beam into your eyes Protect others from exposure to the beam A site preparation and safety guide is a separate document sent to all customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Chemical Hazards Waste Profiles and Disposal Chemical Hazard HWW te CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems Warning instruments and protocols are potentially hazardous and can cause injury illness or death Chemical Waste Hazard Warning Read and understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling
112. ate documents for all entries listed in the Plate ID list saves them to the destination directory adds them to the plate queue and displays a message indicating the number of plate documents it created and sent to the plate queue Click OK to close the message box 8 Click Done The Template Batch dialog box closes 9 From the File menu select Close The SDS software closes the template document 10 Repeat the procedures in this chapter to create and add additional plates to the queue as needed Note Once a plate document has been added to the plate queue the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue 11 When finished creating and adding plate documents to the queue run the queue as explained in Running Plates Using the Automation Controller Software on page 4 34 Run Setup and Basic Operation 4 33 Running Plates Using the Automation Controller Software Launching the The ABI PRISM 7900HT Sequence Detection System employs the Automation Automation Controller Software for automated operation of the 7900HT instrument The software Controller Software coordinates the action of the 7900HT instrument the bar code reader and the plate handler while acquiring and saving raw data during each run To launch the Automation Controller Software as follows Step Action 1 If running the SDS software
113. ate handler arm moves over the input stack 3 Using the Vertical Positioning commands lower the plate handler arm until it is just above the stack The Vertical Positioning box offers four ways to move the plate handler arm Move the slider for large increments Click inside the slider bar to move the arm in 250 step increments Click the lower arrow on the bar to move the arm in 50 step increments Click the up or down arrows in the Vertical Adjustment text box to move the arm in 1 step increments Click the Vertical Adjustment text box type a value and press Enter to move the arm into a specific location Vertical Positioning Slider Slider bar 250 steps per click Down arrow 50 steps per click Vertical Adjustment a Text box arrows 1 step per click 4 Check the rotary position of the plate handler arm to confirm that the gripper is centered over the stack will not contact the sides of the stack when lowered System Maintenance 7 33 7 34 System Maintenance To align the input stack 1 Zymark position 4 continued Step Action 5 Using the Rotary Adjustment arrows adjust the rotational position of the gripper so that it is centered over the input stack and will not contact the sides when lowered To move the plate handler arm clockwise click the up arrow To move the plate handler arm counter clockwise
114. ation component used to analyze the run Raw Spectra Unmodified fluorescence readings taken for each spectral bin during the course of the run When exported the software creates a directory and saves each the raw spectra data for each well in a separate txt file Results Table The contents of the results table of an analyzed plate document Note The contents of the exported data varies depending on the type of plate document Setup Table The contents of the results table of a plate document prior to analysis The contents of the exported data varies depending on the type of plate document used to produce it See page B 4 for a detailed description of the Setup Table file Importing and Exporting Plate Document Data B 9 To export an element of a plate document continued Step Action 5 Select the appropriate well radio button as follows To export data from Select the all wells on the plate document All Wells radio button selected wells of the plate grid only Selected Wells radio button From the files of type drop down list select the appropriate format for the exported data Click the File name text box and type a name for the exported file Click Export The software saves the exported data to the designated location B 10 Importing and Exporting Plate Document Data Designing TaqMan Assays In This Appendix Th
115. ation of each contaminated well Decontaminate the sample block as explained in Decontaminating the Sample Block on page 7 11 Run a background plate to confirm that the contaminants have been removed If the contamination is present after running the background plate for a second time the background plate is likely to be the source of contamination Troubleshooting 8 9 Pure Dye Runs Pure Dye Troubleshooting Table 8 10 Troubleshooting Observation Possible Cause Recommended Action Software will not extract pure dye data During plate setup the wrong plate type was assigned to the plate document Create and run a new pure dye plate document with the proper plate type setting A background plate was not run before the pure dye plate Run a background plate then run the pure dye plate again Raw data from pure dye run appears strange see below Raw Data Plot Calibration Data 3 00 E 4 2 50 E 4 2 00 E 4 1 50 E Amplitude 1 00 E Temperature C a o Pure dye plate was loaded backwards Raw Data Plot 17 Ime a Verify the pure dye wavelengths are as expected b Rerun the pure dye plate Signals plateau saturation Signal is too low lt 10 000 FSU Intensity is set too high low Call Applied Biosystems Technical Support More than two outliers per dye in a single row Evaporation Contamina
116. bar click the Analyze button The software analyzes the run data and displays the results in the Dissociation Curve tab Determine the T for the dissociation curves displayed within the Dissociation Curve tabbed page as explained on page 6 23 Real Time Analysis 6 21 Determining T Values for the Analyzed Run Viewing Analyzed The SDS software displays the results of the dissociation curve analysis within the Dissociation Curve Dissociation Curve tab of the plate document The tab displays the analyzed data ina Data graph of the negative of the derivative R versus temperature 7 that visualizes the change in fluorescence at each temperature interval during the ramp 6 22 Real Time Analysis Note The plot displays data from the selected wells of the plate grid If you do not see dissociation curve data select the wells of the plate grid containing the SYBR Green reactions The following figure illustrates the components of the Dissociation Plot Setup Instrument Results Dissociation Curve Dissociation Curve tab 5 000 E 1 4 000 E 1 3 000 E1 Derivative 2 000 E 1 1 000 E 1 0 000 Detector SYBR Dissociation Plot By Dissociation Plot Dissociation curves Tm display and slider Temperature Plot Derivative Step Stage 6 step 1 f Step drop down list Plot drop down list Detector drop down list The following table describes the el
117. bleshooting Analyzed Real Time Run Data continued Analysis View Description What to look for Amplification Plot Displays data from real time runs after signal normalization and Multicomponent analysis It contains the tools for setting the baseline and threshold cycle C values for the run Correct baseline and threshold settings Are the baseline and threshold values set correctly Identify the components of the amplification curve and set the baseline so that the amplification curve growth begins at a cycle number that is greater than the highest baseline number IMPORTANT Do not adjust the default baseline if the amplification curve growth begins after cycle 15 Identify the components of the amplification curve and set the threshold so that it is Above the background Below the plateaued and linear regions Within in the geometric phase of the amplification curve Irregular amplification Do all samples appear to have amplified normally The three phases of the amplification curve should be clearly visible in each signal Outlying amplification When the run data is viewed in the C vs Well Position plot do replicate wells amplify comparably Wells producing C values that differ significantly from the average for the associated replicate wells may be considered outliers If a plate produces non uniformity between replicates some samples on the plate could have evapora
118. bution of each fluorescent dye used in the sample through a process called multicomponenting see page A 5 The software accomplishes the separation by comparing the raw spectra with a set of pure dye standards contained within a calibration file When a plate document is saved after analysis the software stores the pure spectra information with the collected fluorescent data for that experiment System Maintenance 7 17 Materials Required The following materials are required to perform a pure dye run Material Part Number Sequence Detection Systems Spectral Calibration Kit 384 Well Version 96 Well Version See page D 3 Pure Dye Platea Product Insert Centrifuge with plate adapter a The 96 Well version of the Spectral Calibration Kit contains 2 Pure Dye plates b Included with the Sequence Detection Systems Spectral Calibration Kit Preparing a IMPORTANT A background run must be performed prior to running a pure dye plate See Performing a Background Run on page 7 13 for more information Pure Dye Plate Document 7 18 System Maintenance To prepare a plate document for the pure dye run Step Action 1 Remove the pure dye plate from the freezer place it aside to thaw to room temperature and return to the computer Launch the SDS software From the File menu select New The New Document dialog appears 4 Sel
119. by importing the plate document setup information from a tab delimited text file See Importing Plate Document Setup Table Files on page B 2 for more information Dissociation A dissociation curve analysis is performed as part of a real time PCR run absolute Melting Curve quantification To perform a dissociation curve construct a plate document for Checklist absolute quantification as explained on page 4 3 and configure the method with a temperature ramp as explained on page 4 16 4 4 Run Setup and Basic Operation Section Plate Document Setup In This Section This section discusses the following topics Topic See Page Step 1 Creating a Plate Document 4 6 Step 2 Applying Detectors and Markers to the Plate Document 4 7 Step 3 Configuring the Plate Document with Detector Tasks 4 11 Step 4 Programming the Plate Document Method 4 13 Step 5 Saving the Plate Document as a Template 4 17 Step 6 Creating a Plate Document from the Template 4 18 Step 7 Applying Sample and Plate Information 4 19 Step 8 Running the Plate on the 7900HT Instrument 4 20 Quick Review Note The following table includes a set of abridged procedures for activating the components
120. ca ae ae ce a aa ea oa E 4 gt PE Position Sample Name Detector Task Ct Quantity Qty mean Qtystddev A1 UNKN5K RNaseP Unknown a A2 UNKN5K RNaseP Unknown A3 UNKN5K RNaseP Unknown A4 UNKN5K RNaseP Unknown A5 UNKN5K RNaseP Unknown AG UNKNS5K RNaseP Unknown AT UNKNS5K RNaseP Unknown AB UNKNS5K RNaseP Unknown Ag UNKNS5K RNaseP Unknow A10 NTC RNaseP NTC A11 STNDRD1250 RNaseP Standard 1250 0 A12 STNDRD2500 RNaseP Standard 2500 0 Add AI3 STNDRD5K RNaseP Standard 6000 0 7 Ald STNDRD10K _ RNaseP Standard 10000 0 Passive Reference ROX A15 STNDRD20K__ RNaseP Standard 20000 0 C In Use LINKNINK RNaceP krna B Table pane Each plate document consists of a grid that corresponds to the wells of a reaction device The grid displays well information depending on the type of plate document The information displayed within the cells of the grid are determined by the Plate Grid properties settings located within the Display Settings dialog box Note For more information on configuring the display settings for the plate grid click a help button f The table pane displays the setup and analysis properties for the plate document in a tabular format The table pane can be exported as a tab delimited text file for use by a spreadsheet application see Appendix B Importing and Exporting Plate Document Data for more information Getting Started 3 21 Tabbed Pages Plate documents can contain up to six tabbed pages depe
121. ce cccccceeccccccees 2 3 TOIOOTL TL MSC UMC NG ts eee news ohcei reee a Oe Oe oe ee nen g eee or 2 4 COMOUGCT cats t paneer tae Sa eee ae ee ieee tea eae 2 7 Bal Ode Road iS esasan Sats 8 Ala Rw RET ARAM ss I REA Rae 5 Rede 2 8 Zymark Twister Microplate Handler 20 0 0 ccc eens 2 9 Compatible Consumables e ceirios eni per pa eh aehe bees hansen ee bes 2 10 Instrument COnnecHons ereere sone iat ce Meee oo oo ER PRS eee eae 2 11 Section Getting to Know the Software ccc cece cee eee ence ee eeeees 2 13 Sequence Detection System Software Components and Features 2 14 Managing Sequence Detection System Data 0000 2 15 Working with SDS Software Files 0 00 00 0c ccc eee eee 2 16 Getting Started MN Sale ds gre eaten arabes Gre cake tee buyer Gaede e h Ga E A ee ea en alee eb 3 2 About Thais Mantak pares eA BRM GAS ee Oe dn ele Bree eee ea koe es 3 3 Turning on the ABI PRISM 7900HT Sequence Detection System 3 5 Using the SDS Sotware WorkSpace 24 io ce Wed tade ati saseesme ieee eked os 3 7 Basic Sor ware Skills Tutorials ancvarc dae reon ren bead Guede erie aa ha 3 11 Using SDS Plate DOCUMEMS 5c 2 4 42 a4 Ale aah wh ded Sale Seah be Sees EOS 3 21 4 Run Setup and Basic Operation Belo YoU Beo Baa eis hie a eae ba ee aea aa Se aia eS eee 4 2 Se UPE DECKS 5 4 nrd wine dat mw Pee ce ti ea ele hb et ees Chk net aes wae 4 3 Section Plate Document Setup ccc cece c
122. ce of the reporter Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye The figure below shows the forklike structure dependent polymerization associated 5 3 nuclease activity of Ampli Taq Gold DNA Polymerase during PCR Polymerization R Reporter Q Quencher ae Rm O eee 5 3 Reverse Primer Strand displacement D 5 3 C a a S 5 3 Cleavage A oS 5 Probe 3 3 5 i Polymerization completed R q Probe g When the probe is intact the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by F rster type energy transfer F rster 1948 Lakowicz 1983 During PCR if the target of interest is present the probe specifically anneals between the forward and reverse primer sites The 5 3 nucleolytic activity of the AmpliTaq Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target The probe fragments are then displaced from the target and polymerization of the strand continues The 3 end of the probe is blocked to prevent extension of the probe during PCR This process occurs in every cycle and does not interfere with the exponential accumulation of product The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR Because of these
123. charged coupled device array See instrument optics system checklists absolute quantification 6 7 allelic discrimination 5 8 dissociation curve 6 20 plate document setup absolute quantification 4 3 allelic discrimination 4 4 dissociation curve 4 4 chemical safety 1 3 Index 2 chemistry non optimized 8 5 SYBR Green A 3 TaqMan A 2 troubleshooting 8 4 to 8 7 cleaning gripper finger pads 7 43 sample block wells 7 12 closing instrument tray from the Automation Controller software 4 38 from the SDS software 4 27 plate documents 3 14 Zymark Twister software 7 39 comments adding to a detector 4 7 adding to a plate document 4 19 computer about 2 7 hard drive partitions 2 7 maintaining 7 46 minimum system requirements 2 7 troubleshooting 8 14 turning ON 3 5 configuring the 7900 instrument for 9600 emulation 4 14 consumables 384 Well Optical Reaction Plates D 3 96 Well Optical Reaction Plates D 3 improper or damaged plastics 8 7 Optical Adhesive Covers D 3 Optical Cap Strips D 3 writing on reaction plates 8 6 contamination decontaminating the sample block 7 11 fluorescent common sources 8 6 isolating on the sample block module 8 8 contextual menus using 3 20 copying detectors to a plate document 4 8 markers to a plate document 4 10 Cover readout from the Real Time tab 4 25 creating detectors 4 7 markers 4 9 plate documents 3 12 4 6 for running background plates 7 14 for running custom pure dyes 7 22 for running pure dye
124. click the down arrow Rotary Positioning TE ee Up arrow moves the arm clockwise J Down arrow moves the arm counter clockwise 6 Using the Vertical Positioning commands carefully lower the plate handler arm into the stack Adjust the Rotary Adjustment value as needed to center the gripper within the stack 7 Once the gripper is centered within the stack click Find Plate The plate handler arm lowers upon the plate Confirm the following the plate is in the middle of the gripper span the plate sensor switch is contacting the plate the gripper or plate do not contact the side of the stack 8 Click Close Gripper The gripper grips the plate between its fingers 9 Select Vertical Home The plate handler raises the arm to its highest position If the plate contacts the sides of the stack re adjust the rotary position of the plate handler arm until the plate moves freely in the stack Note Contact between the plate and the stack or all stacks may be unavoidable However try to minimize the contact as much as possible 10 Using the Vertical Positioning commands raise and lower the plate handler arm several times to check the alignment 11 Lower the plate handler arm to the bottom of the plate stack click Rotary Offset and click Yes The software records the rotary position for the Zymark position 4 input stack 1 12 Click Open Gripper The gripper releases the plate Aligning the Plate The next s
125. code P N 4309849 96 Well Optical Reaction Plates 4306737 ABI PRISM 96 Well Optical Reaction Plate with Barcode 20 Plates code 128 4326659 25 Pack ABI PRISM 96 Well Optical Reaction Plate with 500 Plates Barcode code 128 Includes 25 ABI PRISM 96 Well Optical Reaction Plate with Barcode P N 4306737 4314320 ABI PRiSM 96 Well Optical Reaction Plate with Barcode 100 Plates code 128 and ABI PRiSM Optical Adhesive Covers 100 Covers Includes 100 ABI PRISM Optical Adhesive Covers P N 4311971 and 5 ABI PRISM 96 Well Optical Reaction Plate with Barcode packages P N 4306737 Miscellaneous 4312063 MicroAmp Splash Free Support Base for 96 Well Reaction 10 Bases Plates Kits Reagents and Consumables D 3 Instrument The following sequence detection kits and reagents are used to perform routine Maintenance and Maintenance on and verify the function of the ABI PRISM 7900HT Sequence Detection Verification System For more information about the use of the kits below see Chapter 7 System Maintenance Part Number Description Quantity Sequence Detection Systems Spectral Calibration Kits 4328639 ABI PRisM 7900HT Sequence Detection Systems 96 Well Spectral Calibration Kit Includes three ABI PRISM Optical 384 Well Reaction Plates one preloaded and sealed Background plate and two preloaded and sealed Spectral Calibration plates containing eight separate dye standards FAM
126. cons 50 0 5 Click the Sample Volume pL text field and type the volume of the reactions to be run on the plate Note Sample Volume refers to the entire contents of any well including buffer blank or any combination of master mix and nucleic acids IMPORTANT All wells on one plate must contain the same reaction volume 6 Choose from the following options If performing an Then absolute quantification run go to Step 5 Saving the Plate Document as a only Template on page 4 17 absolute quantification run add a temperature ramp to generate dissociation with a dissociation curve curve data as explained on page 4 16 Run Setup and Basic Operation 4 15 Programming a Temperature Ramp for Dissociation Curve Analysis To generate the data required to perform a dissociation curve analysis the 7900HT instrument must be programmed to run a temperature ramp in which it slowly elevates the temperature of the samples while collecting fluorescence measurements once every 7 10 seconds see page 6 18 for a detailed explanation To add a temperature ramp to the method for dissociation curve analysis Step Action 1 From the Instrument tab of the plate document click the Thermal Profile tab The software displays the Thermal Profile tabbed page 2 Click Add Dissociation Stage run The SDS software inserts a temperature ramp at the end of the thermal profile
127. ct Overview The ABI PRISM 7900HT Sequence Detection System is a second generation sequence detection system instrument designed for automated high throughput detection of fluorescent PCR related chemistries The instrument is capable of real time end point and dissociation curve analysis of assays arrayed on multiple formats The 7900HT instrument is optimized for use with Applied Biosystems chemistries including those related to nucleic acid quantification and detection The ABI PRISM 7900HT Sequence Detection System is designed to support several PCR related chemistries available from Applied Biosystems and affiliated companies The SDS software features two types of runs to support the variety of applications Run Analysis Description Supported Chemistries End Point Plate Read In an end point run the 7900HT instrument collects a single reading after completing a PCR run After analysis by the SDS software the resulting multicomponent data is used to assess the presence of target sequences in the unknown samples Allelic Discrimination Real Time In an real time run the 7900HT instrument collects data during each cycle of a pre programmed PCR run After analysis by the SDS software the resulting threshold cycle values C are used to establish quantitative relationships between the initial template concentrations of the unknowns and those of the controls Absolute Quantification Dis
128. ct a different marker b Repeat steps 2 to 5 for the new marker c Repeat steps a and b until the alleles for each marker have been called Scrutinizing the Allele Calls To analyze allele types Step Action 1 Verify the calls for the NTC Allele X and Allele Y controls a From the plate grid select the wells containing the No Template Control samples The software highlights the datapoints within the allele plot b Check that the datapoints cluster in the expected position on the plot c If using positive controls repeat steps a and b for the wells containing the Allele X and Allele Y controls Setup Instrument Results Marker ME can aiei R Pl W el al 2 e l g Allele Y controls should cluster here Legend Allele X Allele Y Allele X amp Y E NTC X Undetermined Allele CYP 2C19 2 1 Allele X controls should cluster here No Template Controls should cluster here Allele X CYP 2C19 2 2 2 Designate samples that did not cluster tightly as Undetermined Samples that did not cluster tightly may Contain rare sequence variations Contain sequence duplications Setup Instrument Results Marker GM gt Cal Micie e el W a al Legend Allele X Allele Y Allele X amp Y E NTC X Undetermined Allele Y CYP 2C19 2 1 Undetermined samples End Point Analysis 5 13 5 14 End Point Analysis
129. cument Data B 5 Setup Table Elements continued Number Contents Description Detector Definitions Element numbers 4 to 6 define the detectors that will appear in the well descriptions that follow in a later section The detector definition consists of three sections the declaration of the number of detectors the detector list header and the detector list 4 Number of This line defines the total number of detectors on the plate Detectors Format Number of Detectors lt tab gt number of detectors lt cr gt Example xxx Number of Detectors 5 5 Detectors This line contains the column headings for the Detector Definitions section of the setup table List Header file that make the file easier to edit using a program such as Microsoft Excel Format Detector lt tab gt Reporter lt tab gt Quencher lt tab gt Description lt tab gt Comments lt tab gt Sequence lt cr gt Example Detector ReporterQuencher Description Comments Sequence 6 Detectors The detector list consists of one or more lines displaying the information for each detector List used on the plate document The Detectors List section must contain one line or definition for each detector present on the plate The number of lines in the Detectors List section must be equal to the number defined in the Number of Detectors section see number 4 above Leave blank the Quencher Dye entry for detectors created for the SYBR G
130. d Sequence Detection Data Collection Serial Cable Computer Automation Controller SDS Software SDS files added to and run from the plate queue Raw data saved to SDS files SDS file run individually Hard drive SDS Software Plate document i Data analysis creation Downstream application specific analysis Product Overview 2 15 Working with SDS Software Files Files Used and Created by the SDS Software Average SDS Plate Document File Size 2 16 Product Overview IMPORTANT Never move or delete a SDS software file or folder unless specifically directed to do so by an Applied Biosystems representative or documentation The SDS software includes many different files and folders Some of these are created to store run data and calibration data others are required to run the software File Type Extension Description Plate Document Files ABI PRISMSDS sds A plate document is a virtual representation of a Single Plate consumable plate used to contain samples and reagents during an sequence detection run During the run the software uses the plate document to coordinate the operation of the instrument thermal cycling data collection to organize and store the data gathered during the PCR and to store the results of the downstream analysis of the run data ABI PRISMSDS sdt Tem
131. d in conjunction with the PCR process or other amplification processes The 5 nuclease detection assay and certain other homogeneous or real_time amplification and detection methods are covered by United States Patent Nos 5 210 015 5 487 972 5 804 375 and 5 994 056 owned by Roche Molecular Systems Inc by corresponding patents and patent applications outside the United States owned by F Hoffmann_La Roche Ltd and by United States Patent Nos 5 538 848 and 6 030 787 and corresponding patents and patent applications outside the United States owned by Applera Corporation Purchase of this instrument conveys no license or right under the foregoing patents Use of these and other patented processes in conjunction with the PCR process requires a license For information on obtaining licenses contact the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or The Licensing Department Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 JpegEncoder Licensing Statement The JpegEncoder and its associated classes are Copyright c 1998 James R Weeks and BioElectroMech This software is based in part on the work of the Independent JPEG Group Redistribution and use in source and binary forms with or without modification are permitted provided that the following conditions are met 1 Redistributions of source code must retain the above copyright notice this list of conditions
132. d ome oth ee ie ea SI PARSER CaN whe hoe 6 15 Section Dissociation Curve Analysis 0 ccc ccc ccc ewe eee cece eee eens 6 17 OVEEVIOW to eeraa atid aioe SO ae oe Be eee bh SS Bek ee ee NA 6 18 BerOle VOU DCC x4 faite cas lS oS ele So oh EAS a SESE Sada yee ad 6 19 ANaly SissCHECKIISts4 y bat bad evades ocd Seeks Bei ee eee 6 20 Analy zine the RUN Data cereri amp BB eased eae HM AREA Oe Ree eater ean 6 21 Determining T Values for the Analyzed Run 00 2 eee eee eee 6 22 AeneA YS 1506 i524 oAN SAE Eon te dome ered eee ohare aees 6 24 7 System Maintenance Recommended Maintenance Schedule 0 0 0 ccc eee eee 7 2 Section Maintaining the 7900HT Instrument 0 ccc ccc ee eee wees 7 3 Replacing the SainpleBlOCK parenn eee be aeeon ok OR eR SE Se SS 7 4 Changing the 7900HT Plate Adapter 0 0 0 0 cc cece eens 7 9 Decontaminating the Sample Block 0 0000s 7 11 Performing a Background RUN sss 6 6vendu caddy eewscaewe PENSEE E E 7 13 Perionmnine a Fure Dye Rutics 2c anro a a R AR E AAA N 7 17 Adding Custom Dyes to the Pure Dye Set 20 0 7 21 Verifying Instrument Performance Using a TaqMan RNase P Plate 7 24 Section Maintaining the Plate Handler 0 1 0 0 ccc ccc cc ee eee eees 7 27 Adjusting the Sensitivity of the Plate Sensor Switch 0 00000 eee 7 28 Alionine the Plate Handler i052 sits tart ooh does oes dew awh wi eteG cake 7 32 Aligning
133. d on page 4 14 b Add a temperature ramp to the method for dissociation curve analysis as explained on page 4 16 Run Setup and Basic Operation 4 13 Programming The SDS software is designed to maximize instrument throughput and therefore does Methods for Allelic not provide the option to thermal cycle allelic discrimination plate documents Discrimination Because allelic discrimination experiments are end point runs that do not require data collection during the PCR thermal cycling can be performed on a dedicated thermal cycler and then transferred to the 7900HT instrument for data collection and analysis If you want to thermal cycle the allelic discrimination plates on Then a designated thermal cycler go on to Step 5 Saving the Plate Document as a Template on page 4 17 the 7900HT instrument follow the procedure below Performing Thermal Cycling of Allelic Discrimination Plates on the 7900HT Instrument To perform the thermal cycling and the plate read using the 7900HT instrument run the plate first as a real time plate document and then again as an allelic discrimination plate document as explained below IMPORTANT Follow the procedure below only if you intend to perform the PCR on the 7900HT instrument Otherwise perform the PCR on a dedicated thermal cycler and then transfer the plate to the 7900HT instrument for data collection To conduct allelic discrimination thermal cycling on th
134. d to any Instrument or part which has been a the subject of an accident misuse or neglect b modified or repaired by a party other than Applied Biosystems or c used in a manner not in accordance with the instructions contained in the Instrument User s Manual This Warranty does not cover the customer installable accessories or customer installable consumable parts for the Instrument that are listed in the Instrument User s Manual Those items are covered by their own warranties Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct those failures of the Instrument to meet the Specifications of which Applied Biosystems is notified prior to expiration of the Warranty Period All repairs and replacements under this Warranty will be performed by Applied Biosystems on site at the Customer s location at Applied Biosystems s sole expense Limited Warranty Statement G 1 No agent employee or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation representation or warranty concerning the Instrument that is not contained in Applied Biosystems s printed product literature or this Warranty Statement Any such affirmation representation or warranty made by any agent employee or representative of Applied Biosystems will not be binding on Applied Biosystems Applied Biosystems shall not be liable for any incidental
135. duct Higuchi et al 1992 Higuchi et al 1993 The use of TaqMan probes and reagents further enhances the method by providing sequence specific amplification of multiple targets for comparative or relative quantification The fewer cycles it takes to reacha detectable level of fluorescence the greater the initial copy number of the target nucleic acid Amplification Plot 8 0 a e 2s Phase 3 plateau 2 0 EEE tech 1 0 o HHHH Phase 2 linear n mis AEA 0 10 20 30 40 Cycle Number When graphed in real time on a linear scale normal amplification of PCR product generates a curve similar to the one shown in the figure above This amplification curve consists of three distinct regions that characterize the progression of the PCR Phase 1 Geometric Exponential Detection of the high precision geometric phase is the key to high precision quantitative PCR The geometric phase is a cycle range of high precision during which is characterized by a high and constant amplification efficiency It occurs between the first detectable rise in fluorescence and before the beginning of the Linear phase When plotted on a log scale of DNA vs cycle number the curve generated by the geometric phase should approximate a straight line with a slope The 7900HT instrument typically delivers sufficient sensitivity to detect at least 3 cycles in the geometric phase assuming reasonably optimized PCR conditions Theory
136. e 4 11 4 10 Run Setup and Basic Operation Step 3 Configuring the Plate Document with Detector Tasks About Detector The detectors applied to each well of the plate document must be assigned a task Tasks that defines their specific purpose or function on the plate The SDS software uses the detector task assignments to determine how to treat the data produced by the wells when analyzing the run data Detector tasks vary depending on the type of experiment for which the plate document was created Applying To apply detector tasks to the detectors of the plate document wells Detector Tasks Step Action 1 Using the Ctrl and Shift keys select the wells of the plate grid containing samples for a particular task described in the table below Experiment Task Apply to Allelic Unknown all detectors of wells containing PCR reagents and Discrimination test samples NTC all detectors of negative control wells containing reagents for the PCR but lacking samples Absolute Unknown all detectors of wells containing PCR reagents and Quantification test samples for quantification Standard the appropriate detectors of wells containing PCR reagents and samples of known quantities NTC all detectors of negative control wells containing PCR reagents but lacking samples 2 From the well inspector click the text field in the Task column for each detector entry and select the appropriate
137. e 4 18 4 32 exporting 3 13 importing setup data 3 14 B 2 methods See methods opening 3 13 programming methods 4 13 removing from the plate queue 4 35 running in batches 4 34 individually 4 20 saving 3 12 as a single plate file 4 22 as a template file 4 17 setting Sample Volume 4 15 setting the passive reference 4 12 plate grid about 3 21 eliminating wells from use 6 12 selecting wells 3 17 viewing well information 3 16 zooming 3 18 plate handler 2 9 7 27 aligning 7 32 to 7 39 arm adjustment knob 7 27 gripper 7 27 plate sensor switch 7 27 cleaning the finger pads 7 43 plate stack positions 7 27 replacing the finger pads 7 43 turning ON 3 5 plate queue adding plate documents from the Automation Controller software 4 35 from the SDS software 4 31 using the Template Batch utility 4 32 removing plate documents 4 35 starting 4 38 stopping 4 38 plate stacks loading plates 4 36 4 37 placing in use 4 37 positions 7 27 Plateau Phase of the amplification curve A 8 plates running batches 4 34 individually 4 23 types See consumables plate sensor switch 7 27 adjusting 7 28 to 7 31 plots hiding 3 16 maximizing minimizing 3 15 resizing 3 15 showing 3 16 Post readout from the Plate Read tab 4 25 precision causes of low precision 8 4 to 8 7 preparing master mixes C 4 plate forarun 4 23 primer and probe concentrations optimizing C 4 programming methods for absolute quantification 4 14 for allelic discrimination 4 14 f
138. e 7900HT instrument Step Action 1 Launch the SDS software 2 Create a real time plate document for absolute quantification as described on page 4 6 Note Itis not necessary to configure the plate document with detectors 3 Program the plate document method with the thermal cycling times and temperatures for your protocol as described in Programming the Method for Absolute Quantification below 4 Run the plate using the real time plate document as described on page 4 21 Note Although large the real time file may be helpful in diagnosing and troubleshooting the experiment later if the data from the allelic discrimination run produces unexpected results 5 Go on to Step 5 Saving the Plate Document as a Template on page 4 17 Programming the Note The following procedure describes how to configure only the basic features of the Method for Absolute method thermal cycler conditions sample volume and data collection options To further customize the method for the plate document click the _ button in the Instrument tab and refer Quantification to the online help for instructions on configuring the auto increment and ramp rate values To create a method for the absolute quantification run Step Action 1 From the SDS software click the Instrument tab of the plate document 2 If necessary select or de select the 9600 Emulation check box Note When the 9600 Emulation check b
139. e appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical T AO CHEMICAL HAZARD Be sure to familiarize yourself with the MSDSs before using reagents or solvents You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below To order documents by automated telephone service Step Action 1 From the U S or Canada dial 1 800 487 6809 or from outside the U S and Canada dial 1 858 712 0317 2 Follow the voice instructions to order documents for delivery by fax Note There is a limit of five documents per fax request To order documents by telephone In the U S Dial 1 800 345 5224 and press 1 To order in English dial 1 800 668 6913 and press 1 then 2 then 1 In Canada To order in French dial 1 800 668 6913 and press 2 then 2 then 1 From any other See the specific region under To Contact Technical Support by country Telephone or Fax Outside North America To view download or order documents through the Applied Biosystems web site Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page click Documents on Demand then click MSDS 3 Click MSDS Index search through the list for the chemical of interest to you then click on the MSDS document number for that chemical to open a
140. e as follows a From the Detector drop down list select another detector b Repeat steps 4 and 5 until the baseline and threshold values have been set for each detector 7 Click OK The software closes the dialog box and configures the analysis with new settings Analyzing the Run Once you have configured the analysis options you can analyze the run data During the analysis the software mathematically transforms the raw data to establish a comparative relationship between the spectral changes in the passive reference dye and those of the reporter dye Based on that comparison the software calculates a cycle threshold C for each reaction standard and unknown The software then generates a standard curve for the run by plotting the standard samples on a graph of C versus initial copy number Note See Appendix A Theory of Operation for a detailed description of the SDS software mathematical transformation of real time run data To analyze the run Step Action 1 Select all wells in the plate grid The software outlines the selected wells with a black line 2 Choose one of the following From the Analysis menu select Analyze From the toolbar click the Analyze button Ep The SDS software analyzes the run data and displays the results in the Results tab 3 Set the baseline and threshold values for each detector on the plate as explained on page 6 10 Real Time Analys
141. e document the software re establishes the connection with the 7900HT instrument 4 28 Run Setup and Basic Operation Section Running Multiple Plates Using the Automation Controller In This Section This section discusses the following topics Topic See Page Adding Plate Documents to the Plate Queue for Automated Operation 4 30 Adding a Plate Document to the Plate Queue from the SDS Software 4 31 Creating Plate Documents Using the Template Batch Utility 4 32 Running Plates Using the Automation Controller Software 4 34 Loading Plates onto the Automation Module 4 36 Operating the 7900HT Instrument Using the Automation Controller Software 4 38 After the Run 4 38 Run Setup and Basic Operation 4 29 Adding Plate Documents to the Plate Queue for Automated Operation Automation The first step in configuring the ABI PRISM 7900HT Sequence Detection System for Operation and the automated operation is to add plate documents to the plate queue The plate queue is Plate Queue amp list of plate document files that the Automation Controller Software uses to identify and run associated plates during automated operation By adding plate documents to the queue they automatically become available for use with the Zymark Twister Microplate Handler IMPORTANT Once a plate document has been added to the plate queue the software locks the file preventing any changes from being made to it until the plate document ha
142. e less than optimal precision Factors that can affect precision are described in detail below Factor See Page Improper Threshold Setting 8 4 Imprecise Pipetting 8 5 Non Optimized Chemistry 8 5 Incomplete Mixing 8 5 Air Bubbles 8 5 Splashing PCR Reagents 8 5 Drops 8 6 Writing on the Reaction Plates 8 6 Fluorescent Contamination on the Plates 8 6 Errors 8 6 Contaminated Sample Block 8 7 Improper or Damaged Plastics 8 7 Low Copy Templates 8 7 Use of Non Applied Biosystems PCR Reagents 8 7 Improper Threshold The key to high precision quantitative PCR is accurate detection of the geometric Setting phase The ABI PRISM 7900HT Sequence Detection System typically delivers sufficient sensitivity so that at least 3 cycles of the geometric phase are visible assuming reasonably optimized PCR conditions The SDS software calculates a fixed signal intensity called a threshold which each signal generated from PCR amplification must reach before it is recognized as actual amplification The calculated threshold is an approximation and should be examined and modified as needed Modifying the Threshold In a real time document of the SDS software the threshold can be modified via the Amplification Plot view following analysis of the run data See Setting the Baseline and Threshold Values for the Run on page 6 10 for more information 8 4 Troubleshooting Imprecise Pipetting Non Optim
143. e to confirm the 7 24 proper operation of the sample block If using an automation accessory also perform the following 8 If changing consumable formats adjust the plate sensor switch on 7 28 the plate handler arm for the new plates 9 Align the Zymark Twister Microplate Handler fixed position bar 7 32 code reader for the new plate format Note Itis necessary to align the plate handler to only the Instrument position Zymark position 2 as explained on pages 7 32 to 7 35 10 Align the fixed position bar code reader for the new plate format 7 40 a For example when replacing a 384 well sample block with a 96 well block Materials Required The procedure below requires the use of the following materials 7 4 System Maintenance Material Part Number Replacement Sample Block if replacing the sample block 5 32 inch Hex key necessary only for certain instruments 5 16 inch Hex key some instruments may require a crescent wrench Handling the Sample Block Removing the Sample Block The interchangeable sample blocks are delicate pieces of equipment containing several fragile components that can break if handled improperly The figure below illustrates the correct locations for handling the interchangeable sample block module Circuitry and connections to 7 the instrument Do Not Touch Heat si
144. ece eee n een eeeeeescees 4 5 Step L Creatine a Plate Document cra nwaed io eRe ete bk eek eR Se Oke eee 4 6 Step 2 Applying Detectors and Markers to the Plate Document 4 7 Step 3 Configuring the Plate Document with Detector Tasks 4 11 Step 4 Programming the Plate Document Method 0000055 4 13 Step 5 Saving the Plate Document as a Template 0000055 4 17 Step 6 Creating a Plate Document from the Template 4 18 Step 7 Applying Sample and Plate Information 0000005 4 19 Step 8 Running the Plate on the 7900HT Instrument 4 20 Section Running an Individual Plate 0 ccc ec cece eens 4 2 Saving the Plate Docume ik sd irreais oie cea dae tees ateNeetien eens eee 4 22 Preparing and Running a Single Plate cere ita renere 440 aeanwacedaasdeeks Led 4 23 Operating the 7900HT Instrument Using the SDS Software 4 25 PACTS RUM fc bie sos Be Gide han ass Be Aa ear eee ere oe anne ee eS 4 27 Section Running Multiple Plates Using the Automation Controller 4 29 Adding Plate Documents to the Plate Queue for Automated Operation 4 30 Adding a Plate Document to the Plate Queue from the SDS Software 4 31 Creating Plate Documents Using the Template Batch Utility 4 32 Running Plates Using the Automation Controller Software
145. echnical Support to visit the Applied Biosystems Web Site Note Applied Biosystems service engineers perform regular updates the SDS software during planned maintenance visits On rare occasions when a piece of the SDS software becomes corrupt it may be necessary to re install the software In the event that the software must be re installed observe the following guidelines to re install or upgrade the software Unless instructed to do otherwise remove the SDS software using the uninstall utility Do not delete the program folder from the Program Files directory Install the SDS software under a user login that has administrator privileges on the computer Unless instructed to do otherwise re install the SDS software to the same directory as the previous installation Review all documentation accompanying the new software such as installation notes or user bulletin The updated version of the software may contain new features that require special consideration Troubleshooting In This Chapter This chapter discusses the following topics Topic See Page Troubleshooting Table 8 2 Low Precision or Irreproducibility 8 4 Background Runs 8 8 Pure Dye Runs 8 10 Real Time Runs Quantitative PCR and Dissociation Curves 8 11 End Point Runs Allelic Discrimination 8 13 Software and 7900HT Instrument 8 14 Zymark Twister Microplate Handler and Fixed Position Bar Code Reader 8 17 T
146. ect others from exposure to the beam 20 30 em x Once the gun has read the bar code the software automatically populates the selected text field with the alpha numeric equivalent of the bar code Click OK to close the Document Information dialog box Getting Started 3 19 Lesson 5 Using Contextual Menus Lesson 6 Using Keyboard Shortcuts Lesson 7 Using the SDS Software Online Help 3 20 Getting Started The SDS software features contextual menus as a time saving devices that provide access to the commands for an associated view or pane To access a contextual menu move the mouse pointer over a pane or view of interest and click the right mouse button The menu appears at the location of the pointer Ss __ JH BRB RRR BL Copyvells va Pf tte 8 8 Paste Wells LEB RRR RRB HidePane m l M KMIKIKIKIKIKIKIK Save Grid to Image File x DX DJ Contextual menu N KIKIKIKIKIKIKIK Display Settings o PIKIPIKI RIIKI INIIAI IIIR ILILILILIL IDIKI PIII III All contextual menus provide the following common commands Command Result See Page Hide lt pane or plot gt Hides the pane or view 3 16 Save lt pane or plot gt Opens the Export Graphic dialog box for exporting the B 8 to Image File selected view or pane as a JPEG graphic file Display Settings Opens the display settings dialog box that allows you 4 17 to modify the appeara
147. ect the following options from the menus within the New Document dialog box From Select Assay Pure Dyes Container lt Select the appropriate plate format gt Template For a384 Well Pure Dye Run select 384 Well Pure Dyes Plate sdt For 96 Well Pure Dye Runs select the template matching the Pure Dye plate you intend to run To run Plate 1 containing FAM JOE NED and ROX select 96 Well Pure Dyes Plate 1 sdt To run Plate 2 containing SYBR TAMRA TET and VIC select 96 Well Pure Dyes Plate 2 sdt Note If no templates are available construct a Pure Dye plate document using the product insert from the Sequence Detection Systems Spectral Calibration Kit and the procedure on page 7 22 Remove the pure dye plate from its packaging 6 Click the Barcode text field and scan the bar code number using the hand held bar code reader 7 Click OK The software displays a plate document with the attributes for a pure dye run To prepare a plate document for the pure dye run continued Step Action 8 Save the Pure Dye plate document as follows a From the File menu select Save b From the Files of type drop down list select ABI PRism SDS Single Plate sds c From the Save dialog click the File name text field and choose from the following Plate Format Type 384 Well PureDye_ lt date in MMDDYY format gt For example the file name for a plate run on May 31 2001 would be PureDye_053101
148. ed below Replacing the To replace the finger pad s Finger Pads Step Action 1 Using the Phillips head screwdriver remove the two small Phillips head screws from the fingers on each side of the gripper then remove the fingers Note Move the plate handler arm into any position where it is easy to access the Screws 2 Using a small flat blade screwdriver pry the worn finger pads off the fingers Note The manufacturer recommends replacing all finger pads at the same time 3 Clean any residual adhesive off the fingers using isopropanol Remove a replacement finger pad from the paper backing and place the finger pad on the appropriate finger position Repeat for the remaining finger pads 6 Install the fingers with the fingers pointing down and the finger pads toward the center of the gripper 7 Insert the screws into the fingers and tighten Note The screws do not automatically align the grippers Make sure that the finger pads are making good contact with the plate when the arm grips a plate System Maintenance 7 43 7 44 System Maintenance Section Maintaining the Computer and SDS Software In This Section This section contains the following information Topic See Page General Computer Maintenance 7 46 Maintaining the SDS software 7 48 Note The SDS software is a multicomponent system that must be maintained to ensure optimal operation of the ABI PRISM
149. edure 4 Click Import The software imports the setup table information from the text file and automatically configures the plate document plate grid and setup table with detector detector task marker and sample data Importing and Exporting Plate Document Data B 3 Setup Table File Format Example Setup To guarantee a successful importation of setup table data into a plate document the Table Files imported setup table file must be configured in the correct format for the assay type The following figures illustrate the orientation of information in tab delimited setup table files as viewed in a Microsoft Excel spreadsheet document The numbered elements of the setup table files are explained on page B 5 Example Setup Table File from an Allelic Discrimination Run E AD_setup table xls 2 4 Output Plate Size 3 Output Plate ID Example AD sds Platet oo o S S o 4 Number of Detectors aS ee es es ee ee aoe Ey E 6 CYP 20921 FAM PAR CYP 209 2 Allele 1 Example prof CYP 2092 J J 7 CYP 20922 e ooo PDAR CYP 209 2 Allele 2_ Example proCYP 2092 d T Da o OYP 209 Allele 1 UNKN ICP 2C9 2 Allele 2 UNKN_ 0l ooo OHD CYP 2092 Allele 1 UNKN OJCYP 2C9 2 Allele 2 UNKN Ol po SHDI D CYP 2C9 2 Allele 1 UNKN COC YP 209 Allele 2 UNKN OO HD1 2 CYP 2092 Allele 1 UNKN COICYP 2092 Allele 2 UNKN_ 0l COICYP 209 2 Allele 2 UNKN OJCYP 209
150. efined as the threshold cycle C for the plot Note It may be necessary to adjust the baseline and threshold settings to obtain accurate and precise data For further information on resetting the baseline and threshold settings see Setting the Baseline and Threshold Values for the Run on page 6 10 1250 Threshold setting Threshold cycle C for the 1250 copy well Cycle Number Significance of Beginning with the equation describing the exponential amplification of the PCR Threshold Cycles n m X X 1 Ex where Xn number of target molecules at cycle n so that n gt m Xm number of target molecules at cycle m so that m n Ey efficiency of target amplification between 0 1 n m number of cycles elapsed between cycle m and cycle n Amplicons designed and optimized according to Applied Biosystems guidelines amplicon size lt 150 bp have amplification efficiencies that approach 100 percent Therefore E 1 so that X n n m X 1 1 n m X 2 To define the significance in amplified product of one thermal cycle set n m 1 so that 1 Xo 2 2X X Therefore each cycle in the PCR reaction corresponds to a two fold increase in product Likewise a change in threshold cycle number of one must equate to a two fold difference in initial template concentration Theory of Operation A 11 Importing and Exporting Plate Document Data In This Appendix This appendix discusses the
151. elect ABI PRism SDS Template Document sdt Click the File name text field and type a name for the template Click Save The software saves the template plate document file From the File menu select Close If the software prompts you to save the plate document click No The SDS software closes the template file Create a plate document from the template as explained on page 4 18 Run Setup and Basic Operation 4 17 Step 6 Creating a Plate Document from the Template Options for Creating The SDS software offers two options for creating plate documents from a template file Plate Documents from the Template individually or in batches Option Description See Page Create an individual The procedure below explains how to create a Follow the plate document from single plate document from a template file for procedure the template running a plate By repeating the procedure you below can create as many plate documents as needed Create multiple plate As a faster alternative to the option above the 4 32 documents using the software includes a Template Batch utility that can Template Batch utility simultaneously create multiple plate documents from the template file Creating a Single To create a plate document from the template file Plate Document from a Template Step Action 1 From the File menu of the SDS software select New The New Document dial
152. elow dR R n m dT The software plots the negative of the resulting derivative data on graph of R versus temperature 7 that visualizes the change in fluorescence at each temperature interval The T for the target nucleic acid can be determined from the graph by identifying the maximum for the rate of change displayed as a peak for the appropriate amplification curve Example Results The following figure illustrates a typical dissociation curve from an experiment run to detect non specific amplification in CDNA samples 6 0 Primer Dimer i Main Product 5 0 Tm 74 9 C Tm 80 5 C 4 0 85 90 95 Temperature C The plot above displays the dual amplification peaks typical of primer dimer formation The amplification from the specific product is displayed with a T of 80 5 C while the primer dimer product has a characteristically lower T of 74 9 C Before You Begin Using SDS For specific instructions on any procedure described within this section refer to the Online Help online help accompanying the SDS software To get help at any time during the procedure click a help button located within the dialog box or window in which you are working Examples in This The illustrations and screenshots that appear within this chapter were created from a Chapter plate run to determine the purity of a B actin amplification in unknown samples Each well of the plate contains SYBR Gre
153. ements of the Dissociation Plot Component Description Dissociation Plot The plot displays data from the selected wells in the plate grid Note The properties of the Dissociation Plot are adjustable For more information on adjusting the appearance of the plot click the help button and see the SDS software online help Step drop down Chooses the data displayed within the plot based on the ramp a 86 5 90 0 Ta ISi If a plate document contains data from more than one temperature ramp the Step drop down list allows you to displays the data from each by selecting the position of the ramp in the thermal profile Tm display and The SDS software displays the T below the green slider see above slider There are two definitions for the T value The chemical definition is the temperature at which 50 of the DNA is in a double stranded configuration The mathematical definition is the maximum value for the first derivative curve within a specific peak The following table describes the elements of the Dissociation Plot continued Component Description Plot drop down list Chooses the data displayed within the plot based on the derivative calculation The list offers the following selections Plat Derivative Raw When selected this option plots the normalized reporter fluorescence data R on a graph of fluorescence vs temperature see below left Derivat
154. emical assays designed to amplify different alleles of a common locus The SDS software uses marker information during data analysis to organize and compare the processed run data IMPORTANT Allelic discrimination plate documents must contain at least one marker To create a marker for an allelic discrimination plate document Step Action 1 If the Detector Manager dialog box is open click Done to close it 2 From the Tools menu of the SDS software select Marker Manager The Marker Manager dialog box appears Click Create Marker The Add Marker dialog box appears Click the Enter name of new Marker text field tyoe a name for the new marker and click OK The new marker appears within the Markers text field Apply detectors to the new marker and follows a From the Markers text field click the new marker to select it The software highlights the selected marker b From the Available Detectors text field click a detector that you want to add to the marker The software highlights the selected detector c Click Add Detector The software applies the detector to the marker and displays it below the Marker entry d Repeat steps b and c for the remaining detector x Markers Available Detectors p a i Nie Allele 2 Detectors assigned to CTP ZEST Attete ty xn _ 2 CYP 2C9 2 Allele 2 Marker CYP 209 2 If evaluating multiple loci repeat steps 3 5 to create additional marke
155. en 1 dye forward and reverse primers and genomic DNA known to contain complimentary binding sites Real Time Analysis 6 19 Analysis Checklist Where You Arein The following checklist illustrates your current position in the overall procedure the Procedure Done Step Procedure See Page J 1 Create an absolute quantification plate document 4 6 J 2a a Create detectors for the absolute quantification probes 4 7 b Copy the detectors to the plate document 4 8 3a a Configure the plate document with detector tasks 4 11 NTC Standard and Unknown b Assign quantities to the wells of the plate document that 4 12 contain standards J 4 a Program the method for the absolute quantification run 4 13 b Add a temperature ramp to the thermal profile 4 16 J 5 Choose from the following If running Then a single plate continue to step 7 the first plate inaseries Save the plate document as an ABI PRISM of plates with identical SDS Template Document as explained on assay configurations page 4 17 J 6 Create a plate document from the template created in 4 18 step 5 J 7 Configure the document with sample names and plate 4 19 information J 8 Prepare and run the dissociation curve plate or plates 4 20 9 Analyze the run data 6 21 10 View the results of the dissociation curve analysis 6 22 11 Determine melting temperature T values f
156. ence Amplified Product emission increases in direct proportion to the amount of specific amplified product As the figure on page A 8 demonstrates the graph of normalized reporter R vs cycle number during PCR appears to have three stages Initially R appears as a flat line because the fluorescent signal is below the detection limit of the Sequence Detector In the second stage the signal can be detected as it continues to increase in direct proportion to the increase in the products of PCR As PCR product continues to increase the ratio of AmpliTag Gold polymerase to PCR product decreases When template concentration reaches 10 8 M PCR product ceases to grow exponentially This signals the third stage of R change which is roughly linear and finally reaches a plateau at about 10 7 M Martens and Naes 1989 The progressive cleavage of TaqMan fluorescent probes during the PCR makes possible the correlation between initial template concentration and the rise in fluorescence As the concentration of amplified product increases in a sample so does the R value During the exponential growth stage the geometric phase the relationship of amplified PCR product to initial template can be shown in the following equation C No N 1 E where N is the concentration of amplified product at any cycle N is the initial concentration of target template E is the efficiency of the system and c is the cycle number Theory of Operation A 9
157. ence to normalize the signals from the reporter dyes see page A 6 for more information To set the passive reference dye for the plate document Step Action 1 From the Passive Reference drop down list select the appropriate reference dye R Passive Reference ia emove Roxy 4 Passive Reference drop down list Select the appropriate passive reference 2 Program the method for the run as explained on page 4 13 4 12 Run Setup and Basic Operation Step 4 Programming the Plate Document Method About SDS Methods During a run the SDS software controls the instrument based on the instructions encoded within the method of the plate document Each new plate document except allelic discrimination contains a default method that must be edited for the specifics of the experiment Methods contain the gt gt gt PO Thermal Cycler Conditions Auto Increment Values Ramp Rates Data Collection Options Reaction Volume Setting To create a method for Then absolute quantification program the method as explained on page 4 14 allelic discrimination see page 4 14 dissociation curve analysis Dissociation curves are preformed as part of a real time PCR run absolute quantification Therefore to perform a dissociation curve analysis do the following a Program the method for the absolute quantification experiment as explaine
158. ent can be run you must save it as an ABI PRISM SDS Single Document for Single Plate sds file Plate Operation To save the plate document Step Action 1 From the File menu of the SDS software select Save As The Save As dialog box appears From the Look in text field navigate to and select a directory for the software to receive the new file Click the File name text field and either Type a file name for the plate document file or Type or scan the bar code number for the plate into the text field Note The SDS software does not require that the file name match the bar code of the corresponding plate Click Save The software saves the plate document to the specified directory Run the plate document and associate plate as explained on page 4 23 4 22 Run Setup and Basic Operation Preparing and Running a Single Plate Pre Run Checklist The following tasks must be complete to run a plate on the 7900HT instrument Done Check See Page A background run has been performed in the last month 7 13 A pure dye run has been performed in the 6 months 7 17 The instrument tray does not contain a plate 4 27 IMPORTANT The instrument tray must be empty to begin a run If the instrument tray contains a plate eject and remove it before continuing Plate Requirements See Consumables and Disposables on page D 3 for a complete lis
159. ent dialog box with settings for the new template Drop Down List Select Assay Absolute Quantification Container 384 Wells Clear Plate Template Blank Template c Click OK The software displays a new plate document with appropriate attributes 2 Choose one of the following options Click the Import button from the General toolbar or From the File menu select Import 3 From the Look In text field of the Import dialog box select the Practice txt file created in Exercise 4 4 Click Import The software imports the setup information of the Practice txt file into the plate grid and table of the empty plate document Note For more information on importing and exporting setup table data using the SDS software see Appendix B Importing and Exporting Plate Document Data Lesson 2 Viewing Because plate documents can display setup and analysis data in multiple views and Resizing Panes simultaneously the SDS software has been designed with several navigational devices to help manage the information This lesson will teach you to use the different aids to reduce screen clutter and ensure efficient use of the software Exercise 1 Resizing Panes Views and Plots You can resize the panes views and plots of plate documents by moving the grey lines dividing them horizontally and vertically To illustrate the this feature resize the plate grid pane horizontally as follows
160. er 3 E Fixed position bar code reader and underlying platform covers 7 Push the instrument tray within the instrument and remove the thermal cycler access cover to permit access to the sample block Note The thermal cycler access cover is secured to the instrument by non locking pins and may require force to remove it no tools are required System Maintenance 7 5 To remove the sample block continued Step Action 8 Using a 5 16 inch Hex key turn the sample block locking bolt counter clockwise until it is very loose but still attached to the sample block locking bar Note Some instruments may require the use of an adjustable crescent wrench to loosen the sample block locking bolt Sample block locking bar Sample block locking bolt 9 Loosen the thumb screw securing the sample block locking bar to the instrument chassis may be a 5 32 Hex bolt on some instruments Thumb screw 10 Lift the sample block locking bar up and out of the instrument 11 Remove the sample block from the instrument as follows a Rotate the release lever at the base of the sample block 90 degrees b Being careful of the cooling fins on the bottom of the sample block slide the sample block out of the instrument and place it on a clean level surface 7 6 System Maintenance Replacing the Sam
161. error Also before using a new pipettor tip to serially dispense a master mix wet the tip once by drawing up some of the master mix and dispensing it back into the mix again Chemistries that have not been optimized may be susceptible to inconsistencies To maximize precision and reaction efficiency optimize the primer and probe concentrations of each individual assay used Refer to the TaqMan Universal PCR Master Mix Protocol P N 43804449 for specific information about optimizing probe and primer concentrations for TaqMan related chemistries For maximum precision the PCR master mix must be mixed to uniformity Once all reaction components are added to master mix it should be vortexed for 4 5 seconds before aliquotting it to the wells of the plate Any dilutions performed during the assay should also be vortexed Air bubbles in the wells can refract and distort the fluorescent signals Ideally the reagents would be applied to the wells using a pipetting technique that does not form air bubbles However if a plate does contain air bubbles they can usually be removed by swinging tapping or briefly centrifuging the reaction plate If PCR reagents splash the undersides of the optical adhesive covers the heat from the lid may bake the liquid to the cover and may distort the signal If solashing occurs briefly centrifuge the reaction plate to remove all traces of liquid from the caps Troubleshooting 8 5 Drops Writing on the Reac
162. ettor tips etc The addition of carrier to the sample such as yeast tRNA or glycogen can help prevent these losses increasing the precision and sensitivity of the assay The Applied Biosystems buffer contains an internal passive reference molecule ROX which acts as a normalization factor for fluorescent emissions detected in the samples see page A 6 IMPORTANT Non Applied Biosystems PCR buffers may not contain the ROX passive reference If running non Applied Biosystems chemistry be sure to set the passive reference for your experiment as explained on Setting the Passive Reference on page 4 12 Troubleshooting 8 7 Background Runs Background Troubleshooting Table Observation Possible Cause Recommended Action Software will not extract During setup the wrong Run anew background plate background data plate type was assigned to document with the proper the plate document plate type setting Background is too high See below 2 2500 FSU Background is too high Sample block contamination a Construct and run a new gt 2500 background plate Background plate contamination b See Isolating Sample Block Contamination below Isolating Sample Block Contamination a Fluorescent standard units The measure of amplitude displayed along the Y axis of the Background Plot Signals exceeding 2500 FSU are considered outside the limit of normal background fluorescence and indicates that
163. example spectra demonstrate how the overlapping component dye spectra contribute to the composite spectrum The SDS software multicomponenting algorithm applies matrix calculations to determine the contributions of each component dye spectra Composite spectrum FAM VIC ROX Background MSE ROX FAM VIC Pure Dye components Background 00 0 600 650 from background Wavelength calibration run Theory of Operation A 5 Normalization of Reporter Signals A 6 Theory of Operation The software uses the pure dye spectra generated as part of instrument calibration see page 7 17 to solve for coefficients a b and c in the following equation Measured spectrum a FAM b VIC c ROX d Background MSE where the coefficients a b and c represent each dye component s contribution to the composite spectrum Note The example calculation above assumes that pure dye components exist for three dyes FAM VIC and ROX and for the instrument background After solving for a b c and d the algorithm calculates the mean squared error MSE which measures how closely the collective multicomponent spectrum conforms to the raw spectra The figure below shows a typical display of the contribut
164. fication of nucleic acids using a standard curve method The objective of absolute quantification is to accurately determine the absolute quantity of a single nucleic acid target sequence within an unknown sample The results of an absolute quantification experiment are reported in the same unit measure of the standard used to make them Absolute quantification on the 7900HT instrument is accomplished through the use of the polymerase chain reaction and the fluorogenic 5 nuclease assay see page A 2 During setup standards diluted over several orders of magnitude and unknown samples are loaded onto an ABI PRISM Optical Reaction Plate containing master mix and TaqMan assays targeting a specific nucleic acid sequence The plate is then loaded into a 7900HT instrument which has been configured to perform a real time run During the thermal cycling the instrument records the emission resulting from the cleavage of TaqMan probes in the presence of the target sequence After the run the SDS software processes the raw fluorescence data to produce threshold cycle C values for each sample see page A 10 The software then computes a standard curve from the C values of the diluted standards and extrapolates absolute quantities for the unknown samples based on their C values see below Note See Appendix A Theory of Operation for more information on the fluorogenic 5 nuclease assay real time data collection or the mathematical transfor
165. following topics Topic See Page Importing Plate Document Setup Table Files B 2 Setup Table File Format B 4 Exporting Graphics B 8 Exporting Plate Document Data B 9 Importing and Exporting Plate Document Data B 1 Importing Plate Document Setup Table Files About the Import The SDS software features the ability to import setup table information detector Function detector task marker and sample name layouts into a plate document from a tab delimited text file The import feature is designed to be a time saving device that facilitates the exchange of setup information between other programs and the SDS software Instead of setting up plate documents individually a third party program can be used to construct setup table files which can then be imported into plate documents for use To guarantee a successful incorporation of setup information from a text file to the plate document the file must Be saved in a tab delimited text format Conform to the setup table file formats described on page B 4 Creating and Importation of setup table data into a plate document is accomplished in three major Importing Setup steps Table Data into a Plate Document Creating an Empty Setup Table File The first step in the procedure is to export a setup table file from a blank plate document Note The blank setup table file can be created using a secondary application Such as Microsoft Excel or a text editor so long
166. for the template document e Click Save The software saves the plate document as a template file sdt 6 Run the custom pure dye plate as explained in Preparing and Running the Pure Dye Plate on page 7 19 System Maintenance 7 23 Verifying Instrument Performance Using a TaqMan RNase P Plate When to Perform Purpose of RNase P Runs 7 24 System Maintenance Applied Biosystems recommends running a TagMan RNase P Instrument Verification Plate When changing sample block formats for the first time As needed to verify the function of the 7900HT instrument TaqMan RNase P Instrument Verification Plate is an experiment run to verify the performance of the 7900HT instrument The sealed plate is pre loaded with the reagents necessary for the detection and quantification of genomic copies of the human RNase P gene a single copy gene encoding the moiety of the RNase P enzyme Each well contains pre loaded reaction mix 1X TaqMan Universal PCR Master Mix RNase P primers and FAM labeled probe and template The following figures illustrate the arrangement of standards and samples on the RNase P plate As shown below the RNase P plate consists of 5 columns of template standards 1250 2500 5000 10 000 and 20 000 copies and two unknown populations 5000 and 10 000 copies RNase P Plate Sample Configuration 384 Well 96 Well Materials Required The following materials
167. g a 384 well plate add 20 uL per well If using a 96 well plate add 50 uL per well b Seal the plate using an optical adhesive cover or optical flat caps Briefly centrifuge the background plate Load the background plate into the 7900HT instrument as follows a From the plate document in the SDS software click the Instrument tab b From the lower portion tab click the Real Time tab c From the Real Time tab of the Instrument tabbed page click Open Close d Place the background plate into the instrument tray as shown below Well A1 Position the plate so that the bar code faces towards the y front of the instrument Note The A1 position is located in the top left side of the instrument tray Click Start The 7900HT instrument begins the background run Note Before starting the run the instrument may pause up to 15 min to heat the heated cover to the appropriate temperature When the background run is complete and the Run Complete dialog box appears a Click OK to close the dialog box b Click Open Close and remove the background plate from the instrument tray c Extract the background component as explained on page 7 16 System Maintenance 7 15 Extracting the In this procedure you will extract the calibration values from the background plate Background document Once extracted the SDS software stores the data a
168. he Plate Document Information Optional Note You can apply sample names after the plate has been run but they must be added prior to the analysis of the run data Note The SDS software features the ability to import setup table information detector detector task and sample name layouts into a plate document from a tab delimited text file See Importing Plate Document Setup Table Files on page B 2 for more information Applying sample names to the plate document Step Action 1 From the plate grid of the plate document select the wells containing the first sample 2 Click the Sample Name text field type a name for the sample and press Enter The software labels the selected wells with the new sample name 3 Repeat steps 1 and 2 for all remaining samples Ej Untitled 1 Setup Instrument i M Sample Name Sample Name Name Use Detector Reporter Task Quantity Color text field appears aa c2 Add Remove Copy to Detector Manager Passive Reference ROX H Marker Color 4 Configure the plate document with plate information as explained below To add or edit any plate information Barcode Operator setting or Plate Comments Step Action 1 From the Tools menu of the SDS software select Document Information The Document Information dialog box appears 2 Edit the Barcode Operator or Plate Comments information Note For more information on the feature
169. he figure below illustrates the general configuration of the computer supplied with the ABI PRISM 7900HT Sequence Detection System Monitor power button Computer power button B 82868 AAAA 6688 AAA __ J IMPORTANT The following requirements are valid for Version 2 0 of the SDS software and may change with future revisions of the 7900HT instrument software and firmware Check the release notes accompanying your version of the SDS software for updates To run the SDS software and or to operate the ABI PRISM 7900HT Sequence Detection System a computer must meet the following minimum requirements Component Minimum Requirement Processor Intel Pentium Il processor 400 MHz or faster Memory 256 MB RAM Hard Drive Minimum 25 GB available hard disk space Additional Drives CD ROM drive Operating Systems Microsoft Windows NT 4 0 with Service Pack 6A During installation of the 7900HT instrument the computer hard drive was partitioned to create the following logical drives Partition Size GB Contains C 2 Operating system files Note Applied Biosystems recommends that you do not install programs to the C drive The computer will boot faster if the C drive contains only the operating system D gt 25 SDS 2 0 Software Automation Controller Software Additional Third Party Software ABI PRISM S
170. hich if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices AAAA Indicates a potentially hazardous situation which if not avoided could result in death or serious injury NDIA Indicates an imminently hazardous situation which if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Safety labels are located on the instrument Each safety label has three parts A signal word panel which implies a particular level of observation or action e g CAUTION or WARNING If a safety label encompasses multiple hazards the signal word corresponding to the greatest hazard is used A message panel which explains the hazard and any user action required A safety alert symbol which indicates a potential personal safety hazard See the ABI PRISM 7900HT Sequence Detection System Site Preparation and Safety Guide P N 4317595 for an explanation of all the safety alert symbols provided in several languages The components of the AB PRISM 7900HT Sequence Detection System contain several lasers including an Argon laser within the 7900HT instrument and a low power laser within each bar code reader fixed position and hand held 7 Na Na ite LASER HAZARD Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots Never look directly into the laser beam Remove jewelry and other items th
171. igate to a directory for the software to save the new file b From the Files of type drop down list select ABI PRISM SDS Single Plate sds c Click the File name text field and either Type a name for the plate document file or Type or scan the bar code number of the plate into the text field d Click Save The software saves the plate document the plate document was the software automatically saves the plate saved previously document 3 When prompted click Yes to submit the document to the plate queue Once a plate document has been added to the plate queue the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue Note To release the plate document from the queue launch the Automation Controller Software and remove the plate document from the queue as explained on page 4 35 4 Click OK to close the dialog confirming that the plate document has been added to the plate queue 5 From the File menu select Close The SDS software closes the plate document 6 Repeat the procedures in this chapter to create and add additional plates to the queue as needed 7 When finished creating plate documents run the enqueued plates as explained in Running Plates Using the Automation Controller Software on page 4 34 Run Setup and Basic Operation 4 31 Creating Plate Documents Using the Templa
172. ign the quantity value as 0 To assign more than one detector to a well then repeat the detector definition text blocks for each detector There is no limit to the number of detectors that can appear in a well IMPORTANT All detectors that appear in this section must have been previously defined in the Detector Definitions section elements 4 6 Format for a single Well Well number lt tab gt SDS Sample Name lt tab gt Detector name lt tab gt Detector task lt tab gt Detector quantity lt tab gt Detector name lt tab gt Detector task lt tab gt Detector quantity lt tab gt Detector name lt tab gt Detector task lt tab gt Detector quantity lt cr gt Example for allelic discrimination setup table files 1 Sample 1 CVE eo a5 UNKN O CrP ACIS Zra UNKN 0 2 Sample 2 GYR 2 COZ sl UNKN 0 GIP 2C9 2Z lt 2 UNKN O 3 Example for absolute quantification setup table files 1 Sample 1 GAPDH UNKN 0 2 Sample 2 GAPDH UNKN 0 3 Sample 3 GAPDH STND 20000 4 Sample 4 GAPDH STND 15000 3 Sample 5 GAPDH NTC O Importing and Exporting Plate Document Data B 7 Exporting Graphics Exporting a Plot asa The SDS software can export most panes and plots of the plate document as JPEG JPEG Graphic File Joint Photographic Experts Group graphic files The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser
173. ile holding the plate click Open Gripper and remove the plate Defining the Bottom The Automation Controller Software requires a bottom position value for all stacks of the Stack This value is used to prevent the plate handler arm from colliding or grinding as it moves to the bottom of each stack To find the bottom of the stack Step Action 1 Remove all plates from the instrument and the plate handler arm 2 Place an empty plate into the output stack Zymark position 0 3 From the Zymark Twister Software click position 0 The plate handler arm moves over the output stack 4 Using the Vertical Positioning commands lower the plate handler arm until it is just above the stack 5 Check the rotary position of the plate handler arm to confirm that the gripper is centered over the stack will not contact the sides of the stack when lowered 6 Using the Rotary Adjustment arrows adjust the rotational position of the gripper so that it is centered over the input stack and will not contact the sides when lowered 7 Using the Vertical Positioning commands carefully lower the plate handler arm into the stack Adjust the Rotary Adjustment value as needed to center the gripper within the stack 8 Once the gripper is centered within the stack click Find Plate The plate handler arm lowers upon the plate Confirm the following The plate is in the middle of the gripper span The plate sen
174. install specification of the ABI PRISM 7900HT Sequence Detection System demonstrates the ability to distinguish between 5 000 and 10 000 genome equivalents with a 99 7 confidence level for a subsequent sample run in a single well The following equation verifies the 7900HT install specifications CopyUnk 3 Sc opyUnk4 gt CopyUnk ad 3 OcopyUnk2 where CopyUnk Average Copy Number of Unknown 1 10 000 replicate population Sopyunki Standard Deviation of Unknown 1 10 000 replicate population CopyUnk Average Copy Number of Unknown 2 5000 replicate population Ccopyunk2 Standard Deviation of Unknown 2 5000 replicate population a These values can easily be obtained from the experimental report window Note Upto 6 wells from each replicate group in a 96 well TaqMan RNase P Instrument Verification Plate can be ignored to meet specification Note Upto 10 wells from each replicate group in a 384 well TaqMan RNase P Instrument Verification Plate can be ignored to meet specification Section Maintaining the Plate Handler In This Section This section contains the following information Topic See Page Adjusting the Sensitivity of the Plate Sensor Switch 7 28 Aligning the Plate Handler 7 32 Aligning the Fixed Position Bar Code Reader 7 40 Cleaning and Replacing Gripper Finger Pads 7 46 Automation
175. io ABE EEE EEEES FEN ad ll allel el ell al ell Ee m cE REE EREESE 3 Select an entire column or row of wells using the headers as follows a Click the header for row A to select all wells in the row The software outlines the wells of row A with a black border b Press and hold either the Shift or Ctrl key then click other columns or row headers to select multiple columns ej Practice sds 1 2 3a 4 s e 7 a 910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 AME BERR RRR RRR RRR SLL nn nnnNn E SLE a 4 Select all wells of the plate grid by clicking the top left corner of the plate grid Row A header The software outlines all of the wells in the plate document with a black border Ej Practice sds Button F i 2 s 4 s elre oio 121314151617 18 19 20 21 22 23 24 AMEE EERE ERR RRR RRR RRR REE SPR RRR RRR BER RRR RRR RRR RRR Ree 5 Using the techniques illustrated in steps 1 to 4 practice selecting portions of the plate grid until comfortable using the feature Getting Started 3 17 3 18 Getting Started Exercise 3 Zooming the Plate Grid You can zoom the plate grid to display the well information by clicking the Zoom Grid button El To illustrate this feature Step Action 1 Click the Zoom Grid button Bf and observe how the grid expands to display the well information Ej Practice sds UNEASE UNEASE UNEASE
176. ion of each component spectra for one well Multicomponent Plot FAM component 9 dl D 8000 D VIC component S 4000 ROX component TE 0 Background E component 2 83 MSE S 73 4 E 63 o k 534 0 10 0 30 0 50 6978 1 10 1 30 Time While multicomponenting illustrates absolute change in emission intensity the SDS software displays cycle by cycle changes in normalized reporter signal R The SDS software normalizes each reporter signal by dividing it by the fluorescent signal of the passive reference dye Because the passive reference is a component of the PCR master mix it is present at the same concentration in all wells of the plate By normalizing the data using the passive reference the software can account for minor variations in signal strength caused by pipetting inaccuracies and make better well to well comparisons of reporter dye signal Note For the example above the resulting data from the normalizing is displayed as FAM R and VIC R Real Time Data Analysis Kinetic Analysis The 7900HT instrument can be used to determine the absolute or relative quantity of a Quantitative PCR target nucleic acid sequence in a test sample by analyzing the cycle to cycle change in fluorescence signal as a result of amplification during a PCR This form of quantitative PCR analysis called kinetic analysis was first described using a non sequence specific fluorescent dye ethidium bromide to detect PCR pro
177. is 6 9 Setting the Baseline Before calculating absolute quantification values the baseline and threshold values and Threshold must be set for all detectors present on the plate Values for the Run 6 10 Real Time Analysis To set the baseline and threshold values for the run Step Action 1 Click the Results tab The software displays the contents of the Results tabbed page 2 If the Amplification Plot is not visible click the Amplification Plot button ap The software displays the results of the sequence detection run in an amplification plot of normalized reporter fluorescence R versus threshold cycle C7 3 From the Plot drop down list select Rn vs Cycle cycle number The software plots the analyzed data in the graph of reporter fluorescence versus 4 Identify the components of the linear scale amplification plot and set the baseline so baseline cycle begins after cycle 15 that the amplification curve growth begins at a cycle number greater than the Stop IMPORTANT Do not adjust the default baseline if the amplification curve growth If the amplification plot looks like Then 2 000 E 1 000 E o 4 4 6 8 10 12 14 16 18 20 2F 24 25 28 30 32 34 36 38 40 the amplification curve begins after the maximum baseline cycle 15 Do not adjust the baseline 2 000 E 1 000 E 1 3 254 fod o 2 4 6 8 10 12 14 16 ts 2 22 24 20 28 30 32 34 36 38 4
178. is appendix discusses the following topics Topic See Page Assay Development Guidelines C 2 Design Tips for Allelic Discrimination Assays C 5 Design Tips for Quantitative PCR Assays C 6 Designing TaqMan Assays C 1 Assay Development Guidelines TaqMan Assay To develop custom TaqMan 5 nuclease assays Development Program Step Action See Page 1 Identify target sequence s C 2 2 Design the TaqMan probes and the forward and reverse primers C 3 3 Order reagents C 4 4 Quantitate the concentrations of the probes and primers C 4 5 Prepare the master mix C 4 6 Optimize the primer concentrations C 4 7 Run the assay C 4 Identify Target A target template is a DNA cDNA RNA or plasmid containing the nucleotide Sequence s sequence of interest For optimal results the target template should meet the following requirements C 2 Designing TaqMan Assays The target nucleotide sequence must contain binding sites for both primers forward and reverse and the fluorogenic probe Short amplicons work best Amplicons ranging from 50 150 bp typically yield the most consistent results If designing assays for quantitative PCR see Design Tips for Quantitative PCR Assays on page C 6 for additional recommendations Design Probes and The following sections contain general guidelines for designing primers and probes Primers For specific design tips refer to the app
179. ive When selected this option plots derivative data R on a graph of the derivative vs temperature see below right The derivative data is the negative of the rate of change in fluorescence as a function of temperature The following figures show the plots accessible from the Plot drop down list Setup Instrument Results Dissociation Curve Setup Instrument Results Dissociation Curve Dissociation Plot f Dissociation Plot H 5 000 1 4 000 E1 3 000 E1 Derivative 2 000 amp 1 1 000 E1 0 000 Temperature Temperature Detector SYBR Piot eet iy Step Stage 6 step 1 Detector SYBR gt Plot MAAA Step Stage 6 step 1 7 Raw Plot Derivative Plot Detector Chooses the data displayed within the plot based on detector name drop down list Determining To determine the T value of a melting curve within the Dissociation Plot T Values Step Action 1 Move the mouse pointer over the green T line located on the Y axis line of the plot The mouse pointer becomes a hand 2 Click and drag the T line to the maximum point of the derivative plot of interest The SDS software displays the T for the curve below the T line 0 000 EE Temperature Plot DARA gt Step Stage 6 step 1 1 000 E1 Tm display and slider Detector SYBR Note The apex of the curvature of represents the maximum rate of change in normalized flu
180. ivide diluted standards into small aliquots store at 80 C and thaw only once before use An example of the effort required to generate trustworthy standards is provided by Collins et al Anal Biochem 226 120 129 1995 who reported on the steps they used in developing an absolute RNA standard for viral quantification Pipetting must be accurate because the standards must be diluted over several orders of magnitude Plasmid DNA or in vitro transcribed RNA must be concentrated in order to measure an accurate Asso value The concentrated DNA or RNA must then be diluted 10 1012 fold to be at a concentration similar to the target in biological samples Kits Reagents and Consumables In This Appendix This appendix discusses the following topics Topic See Page Interchangeable Sample Block Modules and Accessories D 2 Consumables and Disposables D 3 Instrument Maintenance and Verification D 4 TaqMan Pre Developed Assays and Reagents D 5 Custom Oligonucleotide Synthesis D 5 Note Part numbers listed within this appendix are for customers within the United States Contact your Regional Sales Office for local Part numbers and prices see Appendix F Contacting Technical Support for a list of telephone and Fax numbers Kits Reagents and Consumables D 1 D 2 Interchangeable The 7900HT instrument features a Peltier based interchangeable sample block Sample Block module based on the technology e
181. ized Chemistry Incomplete Mixing Air Bubbles Splashing PCR Reagents The calculated quantities of target nucleic acid are directly affected by how precisely the template volumes are added to the reaction mixes Other individually added reagents are also affected by pipetting precision such as variable magnesium affects amplification efficiency Using Master Mixes For this reason Applied Biosystems highly recommends using a master mix All common components to a set of reactions should be mixed together and then dispensed to the wells of the plate Sub master mixes can be used to further improve the precision of identical replicates For example instead of pipetting 5 uL of the same template into four replicate wells pipette 20 uL of the template into a sub master mix then divide the sub master mix into four equal parts for amplification When making each master mix add 5 10 additional volume to compensate for pipetting losses Using Pipettors Pipetting precision is also improved by Calibrating and servicing the pipettors regularly Pipetting larger volumes Reducing the number of pipetting steps whenever possible Increasing the consistency of the pipetting method Consult the manufacturer about the correct method of dispensing liquid volumes accurately from the pipettor For example some pipettors are designed to deliver the designated volume at the first plunger stop so blowing out the residue may cause
182. ld Bar Code Reader Note The following procedure explains how to enter the bar code number into the plate document from the Document Information dialog box You can also scan the bar code into the New Document dialog box during plate document creation To enter a bar code number using the hand held bar code reader Step Action 1 From the Tools menu select Document Information The Document Information dialog box appears Click the Barcode text field Ee Document Information Plate Description Operator Barcode CI ick here File Name 384 Well RNaseP Install Plate sdt Plate 1 Instrument Unknown Assay Absolute Quantification ay Last Modified Tue Nov 20 12 59 57 PST 2001 Acquisition started Le lx The name you enter here will appear on reports Acquisition ended The software places the blinking cursor in the text field While holding the hand held bar code reader 20 to 30 cm away from a plate aim at the center of the bar code and press the trigger The scanner emits a sweeping laser beam that appears as a red line on the plate Slowly move the scanning beam slowly across the bar code until the scan gun emits a high pitched tone acknowledging that it has read the code LASER HAZARD Exposure to direct or reflected laser light can burn the retina and leave permanent blind spots Never look into the laser beam Remove jewelry and anything else that can reflect the beam into your eyes Prot
183. leshooting Troubleshooting Software and Computer Problems Software and Computer Problems 8 14 Troubleshooting Observation SDS software will not launch The software crashes freezes the computer or displays an error message Possible Cause Incorrect start up sequence Corrupted software Computer hardware failure Operating System OS corruption Loose bar code reader cable Recommended Action Follow the solutions listed until the symptom goes away 1 a Turn off the 7900HT instrument b Check cable connections c Restart the computer and logon to the computer d Turn on the 7900HT instrument e Launch the SDS software 2 a Restart the computer and logon to your computer b Reinstall the SDS software c Launch the SDS software 3 Contact Applied Biosystems Service for OS problems or if the computer will not boot up at all You may have to reload the OS from the CDs 4 Contact Dell for troubleshooting the computer hardware Communication error Cables are connected incorrectly Check cable connections and COM port setup See Instrument Connections on page 2 11 Thermal cycler errors Sample block module not fully engaged Reseat the sample block module as explained Replacing the Sample Block on page 7 4 Automation Controller Software cannot find a plate document file File not in correct locati
184. lick Task Manager The Task Manager dialog box appears c Confirm that the Twister software has closed by looking for the Twister software entry in the Task list If the software is still running click the software entry and click End Task to exit the software d From the File menu select Exit to quit the Task Manager 13 Replace the covers for the fixed position bar code reader and the underlying platform removed in step 1 on page 7 32 a ABA PRISM Fixed position bar code reader and underlying platform covers System Maintenance 7 39 Aligning the Fixed Position Bar Code Reader Description The fixed position bar code reader must be set so that it automatically scans the plate s bar code as the plate is placed into the instrument tray by the plate handler Preparing the To move the instrument tray to the Out position Instrument for the Step Action Alignment 1 Remove the cover for the fixed position bar code reader AD Ems
185. ll s Note The Sample name text field will display Mixed if multiple wells with different sample names are selected Detector list Lists all available detectors copied to the plate document In Use check box Toggles the activity of the well If unchecked the software eliminates the data from the selected well from all analysis procedures Passive Reference Displays the fluorescent dye used as a passive reference drop down list Marker Inspector Used only for allelic discrimination runs the Marker Inspector shown below appears as the lower half of the Well Inspector and displays all markers available for the plate Use Marker Color Marker Alpha EEE T Marker inspector Run Setup and Basic Operation In This Chapter This chapter discusses the following topics Topic See Page Before You Begin 4 2 Setup Checklists 4 3 Section Plate Document Setup 4 5 Step 1 Creating a Plate Document 4 6 Step 2 Applying Detectors and Markers to the Plate Document 4 7 Step 3 Configuring the Plate Document with Detector Tasks 4 11 Step 4 Programming the Plate Document Method 4 13 Step 5 Saving the Plate Document as a Template 4 17 Step 6 Creating a Plate Document from the Template 4 18 Step 7 Applying Sample and Plate Information 4 19 Step 8 Running the Plate on the 7900HT Instrument 4 20 Section
186. llelic discrimination plates contain positive controls End Point Analysis 5 7 Analysis Checklist Where You Arein The following checklist illustrates your current position in the overall procedure the Procedure Done Step Procedure See Page J 1 Create an allelic discrimination plate document 4 6 J 2a a Create detectors for the allelic discrimination probes 4 7 b Create a marker for each allelic discrimination probe 4 9 pairing c Copy the marker s to the plate document 4 10 J 3a Assign detector tasks to the wells of the plate document 4 11 NTC and Unknown J 4 If you would like to perform thermal cycling of the allelic 4 13 discrimination plate on the 7900HT instrument create a real time plate document for the plate and program it with the method for the allelic discrimination run Otherwise continue to step 5 J 5 Choose from the following If running Then a single plate continue to step 7 the first plate inaseries Save the plate document as an ABI PRISM of plates with identical SDS Template Document as explained on assay configurations page 4 17 J 6 Create a plate document from the template created in 4 18 step 5 J 7 Configure the document with sample names and plate 4 19 information J 8 a Prepare the allelic discrimination plate or plates and 4 20 perform thermal cycling on a designated thermal cycler b Run the allelic discrimination plate or pla
187. lock wells Block Wells 7 12 System Maintenance Step Action 1 Identify the contaminated wells as follows a If not already run perform a background run as explained on page 7 13 b Identify the contaminated wells on the sample block by following the procedure Isolating Sample Block Contamination on page 8 8 Remove the sample block from the 7900HT instrument as explained in Removing the Sample Block on page 7 5 Using the following figure as a guide locate the suspected contaminated wells on the sample block es A Circuitry and connections to AS the instrument Do Not Touch m Well A 1 A aai o Pipet the appropriate volume of 10 bleach solution into each suspected contaminated well of the sample block module For a 96 well sample block pipet 150 uL bleach solution to each well For a 384 well sample block pipet 40 uL bleach solution to each well Allow the sample block to sit for 3 5 min Using a pipet remove the bleach solution from the wells of the sample block Rinse pipet and remove each contaminated well with 3 treatments of deionized water at the appropriate volume for the sample block For a 96 well sample block rinse affected wells with 150 uL deionized water For a 384 well sample block rinse affected wells with 40 uL deionized water Note Absolute isopropanol can
188. lp menu get help for using a specific click a help button located within the dialog box or dialog box plot or feature window in which you are working For More For information about the ABI PRISM 7900HT Sequence Detection System or the SDS Information software Applied Biosystems recommends the following references Title P N ABI PRISM 7900HT Sequence Detection System Site Preparation and Safety Guide 4317595 ABI PRISM 7900HT Sequence Detection System Software Online Help Microsoft Windows Operating System Online Help About This Manual Intended Audience Background Needed Computer Vocabulary and Operations This guide is written for technicians scientists and researchers who will use ABI PRISM 7900HT Sequence Detection System SDS instruments This manual assumes that you are familiar with the following Basic Microsoft Windows operations such as using the mouse choosing commands working with windows and using the hierarchical file system A general understanding of electronic storage devices and data files An understanding of assay preparation and basic laboratory techniques To use the ABI PRISM 7900HT Sequence Detection System you should be familiar with the following basic computer vocabulary and operations Vocabulary and Operations Description Using the mouse Clicking and double clicking selecting and dragging Choosing commands Using menus and drop down lists dialog boxes radio b
189. mations of sequence detection data Standard Curve Plot Cr Quantity LogN initial concentration The figure above illustrates a standard curve generated from a standard RNase P Installation Plate The arrangement of the samples and standards on the plate are shown in Examples in This Chapter on page 6 6 Algorithmic The SDS software can analyze raw data immediately upon completion of absolute Manipulation of Quantification run The term raw data refers to the spectral data between 500 nm to Raw Data 660 nm collected by the Automation Controller Software during the plate read During the analysis the software automatically applies several mathematical transformations to the raw data to generate a more direct measure of the relationship between the spectral changes in the unknown samples Multicomponenting The first mathematical transformation involves the conversion of the raw data expressed in terms of Fluorescent Signal vs Wavelength to pure dye components using the extracted pure dye standards After the dye components have been identified the software determines the contribution of each dye in the raw data using the multicomponent algorithm See Multicomponenting on page A 5 for a complete description Setting the Threshold and Calling Cys After multicomponenting the baseline and threshold values must be set for the run see Kinetic Analysis Quantitative PCR on page A 7 for more information The result
190. me Connected and awaiting a command ol Disconnected Not connected or turned off 3 10 Getting Started Basic Software Skills Tutorial About This Tutorial Lesson 1 Using Plate Documents This tutorial will Teach you to create save print export and import SDS plate documents Familiarize you with the basic components of the SDS software interface Explain how to customize and arrange the user interface to suit your needs Teach you to use the hand held bar code reader gt gt gt DH Provide you with time saving devices to increase your effectiveness on the SDS software Using the Online Version of the Basic Skills Tutorial The SDS Online Help features a version of this tutorial If you prefer to follow the online tutorial open the SDS Online Help as follows Step Action 1 If not already active launch the SDS software as explained on page 3 7 The SDS software workspace appears From the Help menu select SDS Online Help When the SDS Online Help appears select Basic Skills Tutorial from the list of options 4 Follow the directions displayed on your screen Every plate run on the ABI PRISM 7900HT Sequence Detection System requires the creation of a plate document within the SDS software A plate document is a virtual representation of a consumable used to contain samples and reagents during a sequence detection run The software uses the plate document to coordinate the
191. ments is typically to produce a qualitative result End point analysis is commonly used in combination with TaqMan chemistry to confirm the presence or absence of specific target nucleic acid sequence in cells tissues or fluid samples Currently the SDS software supports one type of end point analysis Allelic Discrimination Section Allelic Discrimination In This Section This section contains the following information Topic See Page Overview 5 4 Before You Begin 5 7 Analysis Checklist 5 8 Analyzing a Completed Allelic Discrimination Run 5 9 Calling and Scrutinizing Allelic Discrimination Data 5 10 After the Analysis 5 15 End Point Analysis 5 3 Overview Allelic The ABI PRISM 7900HT Sequence Detection System supports allelic discrimination Discrimination on Using TaqgMan probes Allelic discrimination is the process by which two variants of a the 7900HT_ single nucleic acid sequence are detected in a prepared sample Allelic discrimination Instrument Chemistry can be used for single nucleotide polymorphism SNP detection Employing the Allelic discrimination on the 7900HT instrument is made possible through the use of 5 Nuclease Assay for the fluorogenic 5 nuclease assay see page A 2 During the PCR the fluorogenic Allelic Discrimination probes anneal specifically to complementary sequences between the forward and reverse primer sites on the template DNA Then during extension AmpliTag
192. mitted by the sensor switch is very faint and may be difficult to hear To make the adjustment easier place your ear close to the sensor switch while making the adjustment and listen for the switch to engage To adjust the plate sensor switch continued Step Action 4 Remove the plate and listen for the plate sensor switch to disengage Did you hear the switch disengage Then No a Move the switch Down a few steps by turning the thumb wheel in the direction indicated on the arm b Replace the plate within the gripper and listen for the switch to engage If you do not hear the switch engage then remove the plate and repeat steps a and b above If you hear the switch engage remove the plate and continue to step a below Yes a Move the switch Up by turning the thumb wheel one step in the direction indicated on the arm b Replace the plate and listen for the switch to engage If you hear the switch engage remove the plate and repeat steps a and b If you do not hear the switch engage then you have successfully identified the zero point of the plate sensor switch Note At the zero point one step of the thumb wheel in the Down direction causes the switch to engage 5 Once the zero point is established carefully turn the thumb wheel in the Down direction the number of steps appropriate for your plate format as indicated below Plate Format T
193. n phase the SYBR Green 1 Dye begins binding to the PCR product Polymerization Complete 3 a eN a a a 5 SAANG aaia TT ci N ae Tar 5 ee UT re rT Polymerization is complete and SYBR Green 1 Dye is completely bound resulting in a net increase in fluorescence Theory of Operation A 3 Fluorescence Detection and Data Collection Fluorescent During PCR light from an argon ion laser is sequentially directed to each well on the Sequence Detection microplate The light passes through the ABI PRism Optical Adhesive Cover and the laser excites the fluorescent dyes present in each well of the consumable The resulting fluorescence emission between 500 nm and 660 nm is collected from each well with a complete collection of data from all wells approximately once every 7 10 seconds A system of lenses filters and a dichroic mirror focus the fluorescence emission into a grating The grating separates the light based on wavelength into a predictably spaced pattern across a charge coupled device CCD camera The SDS software collects the fluorescent signals from the CCD camera and applies data analysis algorithms Charged coupled device array Camera lens an Grating eal o Emission filter E Laser source Beam splitter L Fresnel Lens within Lensplate 384 96 Well Side View Front View A 4 Theory of
194. nce of the view pane or plot The SDS software features keyboard shortcuts for invoking the major functions of the software Exercise Closing the Plate Document To illustrate the use of a keyboard shortcut close the plate document as follows Step Action 1 Simultaneously press the Ctrl and W keys Ctrl W 2 When prompted to save the plate document click No Note The SDS software online help contains a complete list of the keyboard shortcuts for the SDS software To view the list open the online help as explained below The SDS software features an online help system that can guide you through the procedures for setting up performing and analyzing runs To get help at any time click a Help button located within the dialog box or window in which you are working The SDS software provides two ways to access the online help as follows To Then access general help select SDS Online Help from the Help menu get help for using a specific click a help button located within the dialog box or dialog box plot or feature window in which you are working Using SDS Plate Documents Using Multiple Plate Documents Elements of a Plate Document Plate Grid Table Pane The SDS software can handle multiple documents simultaneously however the processing speed of the computer will decrease with each open document For that reason Applied Biosystems recomme
195. nding on their function Tab Used to Setup display well information and to configure the plate grid with setup information Instrument program the plate document method and to run the plate document or send it to the Plate Queue Raw Data display the raw fluorescence collected from the sequence detection run Calibration display the Background and Pure Spectra calibration data used for the signal Data normalization and multicomponenting analysis of the current run Results display analyzed run data The Analysis tab is visible only in plate documents containing analyzed run data Dissociation display analyzed dissociation curve data from a programmed ramp The Curve Dissociation Curve tab is visible only in plate documents containing analyzed data from a real time run with a programmed ramp Well Inspector The Well Inspector is used to apply detector and sample information to the wells 3 22 Getting Started within the grid pane and to display information from the selected cells in the plate grid Setup instrument Sample Name z Sample name text field Use Detector Reporter Task Quantity Color a RNaseP FAM Mixed o m Detector list Passive Reference drop down list Passive Reference Add Remove M In Use In Use check box Component Description Sample name An editable text field that displays the sample name applied to the text field selected we
196. ndler arm will grasp the plate and remove it from the plate stack If plate handler arm stops before the gripper fingers are able to contact the plate and fails to grasp or pick up the plate adjust the plate sensor switch as follows a Turn the thumb screw in the Up direction 10 steps b Grasp the plate with one hand and from the Zymark Twister Software click Open Gripper to release the plate c Replace the reaction plate into input stack 1 of the plate handler d Repeat steps 6 and 7 until the plate handler arm successfully retrieves the plate Grasp the plate with one hand and from the Zymark Twister Software click Open Gripper to release the plate 7 30 System Maintenance To test the sensitivity of the plate sensor switch continued Step Action 9 Exit the Zymark Twister Software a Click Main Menu The software displays the main menu b Click Quit Application The software closes 10 Note A bug inthe Zymark Twister Software can cause portions of the program to persist in memory even after the software has been closed Because the Zymark Twister Software conflicts with the SDS software the residual elements of the software must be closed within the Windows Task Manager before continuing Confirm that the stack has closed by viewing the Task Manager a Press the Crtl Alt Del keys in unison The Windows Security dialog box appears b Click Task Manager The Task Manager
197. nds limiting the number of open documents to 10 This section will familiarize you with the elements of plate documents The figure below illustrates a typical plate document The following pages describe its components fj Practice sds BEE HOER E ESEA 8 9 10 1 12 13 14 15 16 17 18 19 20 21 22 23 24 Setup instrument Tee Cee eee eee eee o Well inspector E Ri O Ri O Ri O ei O ei A ri A Ri A Ri A Ri T Ri ET Ri E Ri E Ri E Ri E ei a ei M ei O Ri I Ri A Ri I Ri T Ri M Ri Sample Name bE E E EEEE EERE oe eo oe ee oe he he he Ue he E E E M Ri D Ri D Ri M Ri MN Ri a Ri MN Ri MN pi AA ei E ei E ei E ei E ei E ei D ri D Ri D Ri D Ri I Ri D Ri D Ri M Ri M Ri Use Detector_ Reporter_ Task _ Quantity _Color_ BREREBRBHREHBRHEHEHEHEEBEHEHBEHEEBEHEHEHEEHESBEESBE SB SB m Raser Stan O E ee M ri M ri M ri M ri M ri M ri M ri A oroi Se A r A nr A r B ri M ri M ri M ri M ri M pri M ri M ri M ri M ri DE EEE EEEE EEEE es es ee et eh Ue Ue he MM Rri M a a ri M ri M r M ri M r Be Se Se A nr A r B ri M ri M ri M ri M ri M pri M ri M ri M ri M ri cE EEEE OO Oe oe oe Oe oe oe oe he he oe ho oe ho NN E I I ee OO Ri O Ri I ES Ri E Ri E Ri E Ri es D Ri D Ri O pi M es O Ri FE EEEE EERER EERE ERE EREEREER EEEAEEEEEE Nn m ri M ri M ri M ri M ri M ri M ri M r M nr M oi A nr A rR A nr A nr A nri M ri M ri M nr M ri M ri M ri M ri M ri M ri oHm E Os OO oo oe ee oe oe oe Ri M ri M ri M ri M ri M Ri M
198. ng analysis options for absolute quantification 6 8 display settings 4 17 method step parameters 4 15 plate sensor switch 7 28 to 7 31 adjustment knob 7 27 air bubbles 8 5 aligning fixed position bar code reader 7 40 to 7 42 plate handler 7 32 to 7 39 allele calls about 5 12 calling 5 11 scrutinizing 5 13 allelic discrimination about 5 4 to 5 6 analyzing run data 5 8 to 5 15 assay development guidelines C 5 maximizing throughput 4 2 procedure checklist 5 8 setting up procedure checklist 4 4 thermal cycling on the 7900HT instrument 4 14 troubleshooting 8 13 Allelic Discrimination Plot about 5 10 calling alleles 5 11 datapoint cluster variations 5 5 exporting as a graphic file B 8 exporting data as atext file B 9 genotypic segregation 5 6 Outliers 5 6 scrutinizing allele calls 5 13 amplification curve about A 7 Geometric Exponential Phase A 7 Linear Phase A 8 Plateau Phase A 8 Amplification Plot exporting as a graphic file B 8 exporting data as atextfile B 9 setting the baseline 6 10 setting the threshold 6 11 visualizing outliers 6 12 analyzing absolute quantification data 6 7 to 6 14 allelic discrimination data 5 8 to 5 15 background data 7 16 dissociation curve data 6 20 to 6 24 pure dye data 7 20 applying detector tasks 4 11 detectors to a plate document 4 8 markers to a plate document 4 10 sample names 4 19 archiving SDS files 7 46 assay development guidelines absolute quantification C 2 to C 4 C 6 allelic
199. ng Started Using the General The following table describes the icons located within the General Toolbar Toolbar Icon Function Creates a new plate document Opens an existing plate document Saves the current plate document Imports data from a text file Exports data to a tab delimited text file Opens the SDS software Find utility Removes the selected object and places it into memory Copies the selected object into memory Inserts a cut object into the current selection Ly ee Le les 1 ee B Ie Analyzes the current plate document LI Opens the Analysis Options dialog box for the current plate document Using the Display The following table describes the icons located within the Display Toolbar Toolbar Icon Function Hides or shows the Well Inspector Panel Hides or shows the Plate Grid Hides or shows the Table View Hides or shows the System Raw Data Plot Hides or shows the Multicomponent Plot Hides or shows the Amplification Plot Hides or shows the Standard Curve Plot Hides or shows the Dissociation Plot Zooms the plate grid in or out 3 e E eg ig E e i es a Opens the Display Settings dialog box used to modify the appearance of the plate document plate grid plots and views Getting Started 3 9 About the Status Bar The status bar consists of two components a message bar for indicating the s
200. ning or closing dialog boxes and other software processes Hard drive is fragmented Defragment the hard drive as explained on Defragmenting the Hard Drive on page 7 47 Hard drive is almost full Remove or archive existing data files as explained on Archiving SDS Files on page 7 46 The computer will not logon to the Windows Operating System Logon window does not appear Restart the computer and logon to your computer You are not logged on as the Administrator a Logoff of your computer b Logon again as the Administrator After the above solutions have been tried the problem is still not fixed Contact Dell for troubleshooting the computer hardware or OS Troubleshooting 8 15 8 16 Troubleshooting Troubleshooting Software and Computer Problems continued Observation The computer will not boot up at all Possible Cause Cables are not connected or are not seated properly Recommended Action Check the cables The boot disk is corrupted a Boot directly off of the NT Installation CD b Boot off of the emergency disk c Reload the Windows NT Operating System from the CD After the above solution has been tried the problem is still not fixed Contact Dell for troubleshooting the computer hardware Zymark Twister Microplate Handler and Fixed Position Bar Code Reader Automation Accessory Troubleshooting
201. nks Bottom of module Hold sample block module from the sides To remove the sample block Step Action 1 Launch the Automation Controller Software and perform the following tasks a Click the Thermal Status tab and confirm the function of the current module The module is operating normally if the software is receiving a temperature reading b Click Open Close to rotate the instrument tray to the OUT position 2 From the File menu select Exit to close the Automation Controller Software Turn off and unplug the 7900HT instrument 7 Ne ea tite PHYSICAL HAZARD The instrument must be unplugged and turned off at all times during the following procedure Failure to comply can result in serious physical injury to the user or damage to the instrument 4 Wait 20 30 min for the heated cover to cool 7 470 PHYSICAL HAZARD During instrument operation the temperature of the sample block can be as high as 100 C Before performing this procedure wait until the sample block reaches room temperature 5 If the instrument tray is in the OUT position outside of the instrument push it into the instrument to provide an open work space 6 If using a Zymark Twister Microplate Handler remove the covers for the fixed position bar code reader and the underlying platform tw D
202. nt and Automation Module Problems Software and 7900HT SDS software will not launch 8 14 Instrument Software crashes freezes the computer or displays an error message Communication error Thermal cycler errors Automation Controller Software cannot find a plate document file Computer and or software displays the Run Completed Successfully dialog box but will not respond and appears to be frozen Run will not start Computer is slow when analyzing data opening or closing dialog boxes and other software processes The computer will not logon to the Windows Operating System The computer will not boot up at all G0 _ h N Zymark Twister Microplate Handler and Fixed Position Bar Code Reader Plate handler emits grinding noise when picking up or putting down plates Plate handler arm contacts racks when retrieving or stacking plates Plate handler arm releases plates awkwardly into the plate stack Reaction plates tip or tilt when placed into the instrument tray by the plate handler arm Plate handler fails to sense or grasp plates Plates stick to the gripper fingers of the plate handler arm Plate handler does not restack plates in original locations Fixed position bar code reader not reading plate bar codes Troubleshooting 8 3 Low Precision or Irreproducibility Overview There are many reasons why an assay run with the ABI PRISM 7900HT Sequence Detection System can hav
203. nt in your data eliminate them as explained below Otherwise go on to Viewing Results on page 6 13 Eliminating Wells from the Analysis IMPORTANT If one or more wells are removed from use before a plate document has been run the SDS software will not collect data for those wells If you identified any outliers in the previous procedure you must eliminate them from the standard curve calculation before viewing the results To eliminate outliers from the calculations Step Action 1 While pressing and holding the Ctrl key click each well in the plate grid that you identified as an outlier in the previous procedure The SDS software creates a black boarder around each cell as it is selected Click the Setup tab From the well inspector of the Setup tabbed page uncheck the In Use check box From the Analysis menu select Analyze O AJOIN Reapply the baseline and threshold values determined on page 6 10 Viewing Results Viewing the Displays the results of the absolute quantification calculation in the results table of the Analysis Table plate document The figure below shows an example of the results table containing the data from a TaqMan RNase P Instrument Verification Plate Posit Sample Name Detector Task Quantity Qty mean Qty stddev Help button Unknown Po RNase P RNase P RNase P RNase P RNase P RNase P RNase P RNase P Unknown Po R
204. nted file systems The software utility defragments broken files by combining their component pieces at a single location on the hard drive thereby optimizing system performance Do not upgrade the operating system of the computer connected to the 7900HT instrument unless instructed to do otherwise by an Applied Biosystems service engineer New versions of the Microsoft Windows operating system can be incompatible with the SDS software and render it and the instrument inoperable The Applied Biosystems service engineer maintains the operating system software as part of planned maintenance visits During the visit the engineer will update the computer operating system as upgrades become available and are validated by Applied Biosystems System Maintenance 7 47 Maintaining the SDS software Administration Privileges Upgrading the 7900HT SDS Software Reinstalling the Software 7 48 System Maintenance IMPORTANT You must have administrator privileges on the computer to install and or upgrade the SDS software Applied Biosystems continually develops the SDS software to provide increased functionality and reliability of the ABI PRISM 7900HT Sequence Detection System As updates become available Applied Biosystems sends notifications of the upgrades to all ABI PRISM 7900HT Sequence Detection System customers If an upgrade is user installable it can be found on the Applied Biosystems company Web site see Appendix F Contacting T
205. ntroller Software adds the plate document s to the Plate Queue Note Once a plate document has been added to the plate queue the software locks the file preventing any changes from being made to it until the plate document has been run or removed from the queue Removing Plate To remove plate documents from the plate queue Documents from the Plate Queue To remove Then specific plate a While pressing and holding the Ctrl key click the plate documents from the document files to remove plate queue The software highlights selected files b From the File menu select Clear Selected Plate s The software removes the selected plate documents from the plate queue all plate documents From the File menu of the Automation Controller Software select from the plate queue Clear All Plates The software removes all plate documents from the plate queue Run Setup and Basic Operation 4 35 Loading Plates onto the Automation Module Pre Run Checklist The following tasks must be complete to run plates on the 7900HT instrument See the associated page number for details on each procedure Done Check See Page A background run has been performed in the last month 7 13 A pure dye run has been performed in the 6 months 7 17 The plate queue contains plate documents for all plates to be run 4 6 The instrument tray does not contain a plate 4 38 IMPORTANT The in
206. nts 2 3 firmware 2 14 internal components 2 4 loading plates 4 24 maintaining 7 2 optics system 2 6 A 4 safety labels 1 2 Status lights 3 6 supported runs and chemistries 2 2 troubleshooting 8 14 turning ON 3 5 instrument tray 4 38 opening and closing from the Automation Controller software 4 38 from the SDS software 4 27 replacing the plate adapter 7 9 irreproducibility causes 8 4 to 8 7 J Java Runtime Environment about 2 14 K keyboard shortcuts using 3 20 L labels 1 2 laser source See instrument optics system launching Automation Controller software 4 34 SDS software 3 7 LAVA software about 2 14 aligning the fixed position bar code reader 7 40 to 7 42 launching 7 40 LDHost software See LAVA software learning to use the SDS software 3 11 lights See instrument status lights limited warranty statement G 1 Linear Phase of the amplification curve A 8 loading plates into the instrument 4 24 into the plate handler stacks 4 37 low copy templates 8 7 Index 4 M maintenance schedule 7 2 manual See user guide marker inspector 3 22 markers about 4 9 applying to a plate document 4 10 copying to a plate document 4 10 creating 4 9 importing into a plate document B 2 master mixes preparing C 4 using 8 5 maximizing instrument throughput 4 2 maximizing minimizing panes views and plots 3 15 melting temperature definition 6 22 determining 6 23 methods about 4 13 to 4 16 adding a hold cycle
207. o select it Note The plate document must be the top most object in the workspace From the File menu select Export Navigate to the directory you would like to receive the exported file s 4 Click the Export drop down list and select the type of data you would like the software to export Data Type The exported file contains Background Fluorescence readings for each well from the background component Spectra used to analyze the run Clipped Average R and AR of the last three data points collected during the extension phase of each cycle repetition for each well Dissociation Temperature Data For each well in use on the plate the file Curve displays the calculated temperature of the wells during each data collection reading of the temperature ramp Raw Data For each well in use on the plate the file displays the R of the well during each data collection reading of the temperature ramp Derivative Data For each well in use on the plate the file displays the first derivative data during each data collection reading of the temperature ramp Multicomponent Calculated amounts the dye components in a single well throughout all stages of the PCR that were labeled with data collection icons Pure spectra component data Calculated inverse matrix Singular values of the inverse matrix Pure Spectra Fluorescence readings for each well from the pure spectra calibr
208. of these datapoints can vary along the horizontal axis Allele X vertical axis Allele Y or diagonal Allele X Allele Y This variation is due to differences in the extent of PCR amplification which could result from differences in initial DNA concentration The example below shows the variation in clustering due to differences in the extent of PCR amplification Seip neta Results cal Aleex z NEN 3 Allele Y homozygotes Allele X Allele Y heterozygotes Legend Allele Allele vy Allele 27 E HTC w Undetermined Allele Y CYP 2C419 2 1 Outliers Allele X homozygotes No amplification 1 0 o 0 1 0 20 3 0 40 Allele x CYP 2C19 2 2 End Point Analysis 5 5 5 6 End Point Analysis Genotypic Segregation of Datapoint Clusters The figure on the previous page illustrates the concept of genotypic segregation of samples within the allele plot The plot contains four separate distinct clusters which represent the No Template Controls and the three possible genotypes allele X homozygous allele Y homozygous and heterozygous Because of their homogenous genetic compliment homozygous samples exhibit increased fluorescence along one axis of the plot depending on the allele they contain In contrast heterozygous samples appear within the center of the plot because they contain copies of both alleles and therefore exhibit increased fluorescence for both reporter dyes
209. of Operation A 7 Determining Initial Template Concentration and Cycle Number A 8 Theory of Operation Phase 2 Linear The linear phase is characterized by a leveling effect where the slope of the amplification curve decreases steadily At this point one or more components have fallen below a critical concentration and the amplification efficiency has begun to decrease This phase is termed linear because amplification approximates an arithmetic progression rather than a geometric increase Because the amplification efficiency is continually decreasing during the Linear phase it exhibits low precision Phase 3 Plateau Finally the amplification curve achieves the plateau phase at which time the PCR stops and the R signal remains relatively constant At any given cycle within the geometric phase of PCR the amount of product is proportional to the initial number of template copies When one template is diluted several times as with the RNase P target in the TagqMan RNase P Instrument Verification Plate see Appendix D the ratio of template concentration to detectable signal is preserved within the exponential phase for all dilutions see below This relationship appears to change as rate of amplification approaches a plateau 8 0 z z z 2 0 1 0 20000 10000 tA 5000 2500 1250 E pL TTT Cycle Number Fluorescence vs When using TaqMan fluorogenic probes with the 7900HT instrument fluoresc
210. og box appears 2 Configure the New Document dialog box as follows Drop Down List Select Assay the same assay as the template Container the same plate type as the template Template the template file sdt created on page 4 17 If the template file does not appear within the Template drop down list select the file as explained below a Click Browse The Open dialog box appears b From the Look in text field navigate to and select the template file sdt created on page 4 17 c Click Open The Template drop down list displays the template file a The Template drop down list displays all template files contained in the Templates subdirectory of the SDS 2 0 program directory 3 Optional Click the Barcode text field and either Type the bar code number for the plate or Scan the plate bar code using the hand held bar code scanner 4 Click OK The software creates a new plate document from the template file 5 Configure the new plate document with sample and plate information as explained on page 4 19 4 18 Run Setup and Basic Operation Step 7 Applying Sample and Plate Information Applying Sample The plate document must contain sample attributes to effectively organize and Names to the Plate analyze data produced from the run Once applied the software displays the sample Document ames within the plate grid and table views Configuring t
211. on Remove file entry from plate queue and add the file to the plate queue again Dialog box does not respond to mouse clicks or key strokes Java Runtime Error Click the close box of the dialog box to close it Troubleshooting Software and Computer Problems continued Observation Run will not start Possible Cause No calibration file No background data in calibration file background run has not been performed No pure dye data in calibration file pure dye run has not been performed Calibration file does not contain pure dye data for a dye used on the plate document Calibration file was created on another instrument Recommended Action Perform background and pure dye runs See Performing a Background Run on page 7 13 and Performing a Pure Dye Run on page 7 17 Disk drive containing the plate document has less than 50 MB of free space Check the capacity of the destination drive If less than 50 MB of free space remains remove or archive existing data files see page 7 46 Heated cover cannot reach running temperature because no plate loaded Instrument tray contains a plate Open the instrument tray and check that the instrument contains a plate Output stack contains a plate or plates Remove all plates from the output stack of the plate handler before starting the queue Computer is slow when analyzing data ope
212. or can be reduced in the following ways Perform the assay in a systematic fashion For example the pattern of sample positions should be simple such as avoid putting gaps in the rows When pipetting the master mix look directly down into the reaction plate so that you can verify the transfer of the solution If adding asmall volume reagent such as template place the drop of liquid on the side of the well Briefly tap or centrifuge the plate afterwards to bring the droplet down into the well After all pipetting is complete visually inspect all the wells to confirm the presence of the reagent drops Tapping or centrifuging the reaction plate will cause all the drops to slide down into the wells simultaneously When making serial dilutions be sure to change the pipet tip after each dilution step Visually inspect the liquid volumes being pipetted to verify that the volume is approximately correct Acommon mistake is using the wrong pipettor volume setting such as setting 20 uL instead of 2 0 uL Visually inspect the volumes of the completed reactions looking for any wells that have volumes that do not match those of the other wells Contaminated Sample Block Improper or Damaged Plastics Low Copy Templates Use of Non Applied Biosystems PCR Reagents Any material contaminating the sample block can affect the results For example mineral oil reduces thermal transfer Residue from writing on reaction
213. or dissociation curve analysis 4 16 temperature ramp 4 16 pure dye plate constructing for custom dyes 7 21 preparing for use 7 19 pure dye run about 7 17 creating a plate document 7 18 extracting data 7 20 performing 7 19 troubleshooting 8 10 when to perform 7 17 Index 5 Q quantifying probes and primers C 4 standards for absolute quantification C 6 quantitative RT PCR about 6 2 absolute See absolute quantification types of 6 2 quantities applying to a plate document 4 12 R R2 readout from the Standard Curve Plot reagents custom oligonucleotides D 5 non Applied Biosystems PCR reagents 8 7 TaqMan Pre Developed Assays and Reagents D 5 TaqMan RNase P Instrument Verification Plates D 4 re connecting the SDS software 4 28 removing outliers 6 12 plate documents from the plate queue 4 35 steps from a method 4 15 wells from use 6 12 Rep readout from the Real Time tab replacing gripper finger pads 7 43 sample block module 7 4 reporter signal normalization A 6 resizing panes views and plots 3 15 restacking plates 4 37 R See normalized reporter signal running batches of plates 4 34 single plate 4 23 6 14 4 25 S safety chemical hazards chemical waste 1 labels safety 1 2 laser exposure 1 waste disposal 1 waste profiles 1 4 sample block locking bar 7 6 sample block locking bolt 7 6 sample block module about 2 5 cleaning sample block module wells 7 12 contamination 8 7 replacing 7 4
214. or the derivative 6 23 peaks 12 Choose from the following post analysis options 6 24 Reanalyze the run data Adjust the display settings for the results table plate grid and plate document plots Print elements of the plate document Export the plate document results table or plots a Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab delimited text file See Importing Plate Document Setup Table Files on page B 2 for more information 6 20 Real Time Analysis Analyzing the Run Data Analyzing the Run The run data from a temperature ramp can be analyzed immediately following the completion of the run For an explanation of how the software manipulates the raw data see Algorithmic Manipulation of Raw Data on page 6 5 To analyze the run Step Action 1 If not already open launch the SDS software as explained on page 4 5 2 Open the plate document file for analysis as follows a From the File menu select Open b From the File Type drop down list select ABI PRism SDS Single Plate sds c From the Look in text field navigate to and select the plate document file d Click Open The SDS software displays the plate document file Select all wells in the plate grid The software outlines the selected wells with a black line Choose one of the following From the Analysis menu select Analyze From the tool
215. ore performing a Pure Dye calibration Pure dye data is generated from the results of a pure dye run in which the SDS software collects spectral data from a set of dye standards during a 2 min hold at 60 C The software stores the spectral information for the pure dye standards within a calibration file located in the SDS directory After the run the software extracts each component dye spectrum from the collected data in the pure spectra run file IMPORTANT Because the age and use of instrument components can affect pure spectra readings Applied Biosystems recommends updating the pure spectra data files once or twice annually depending on instrument use The ABI PRISM 7900HT Sequence Detection System monitors fluorescent signals generated by several dyes FAM NED ROX SYBR TAMRA TET and VIC The figure below compares the pure spectra for each dye Dye Peak nm FAM 520 SYBR 520 3 TET 540 4 VIC 550 JOE 550 5 NED 570 TAMRA 580 ROX 610 Note The 7900HT instrument supports the detection of custom pure dyes dyes other than those provided by Applied Biosystems To add custom pure dyes to the Pure Dye set for your instrument see Adding Custom Dyes to the Pure Dye Set on page 7 21 After a run the SDS software receives run data in the form of a raw spectra signal for each reading To make sense of the raw data the software must determine the contri
216. orescence Real Time Analysis 6 23 After the Analysis Changing the Plate Document Display Settings Saving the Plate Document Printing a Report Exporting Plate Document Data 6 24 Real Time Analysis Before printing or exporting the analyzed data the software allows you to reconfigure the appearance of several elements of the plate document including the results table plate grid and most plots To configure the display settings for the plate document Step Action 1 From the View menu select Display Settings 2 From the Display Settings dialog box click the help button for further instructions on modifying the display settings The software allows you to save any changes to the appearance of the plate document however it does not save the threshold or baseline values made during the analysis To save the plate document select Save from the File menu The SDS software can print a report of the analyzed data containing individual or multiple elements of the plate document To print a report of the plate document data Step Action 1 From the File menu select Print Report 2 From the Print Report dialog box click the help button for instructions on setting up previewing and printing the report Exporting Plate Document Data as a Tab Delimited Text File The SDS software can export raw or analyzed data in tab delimited txt format fo
217. ot or by clicking and dragging a section of the plot to view a Zooms out on the plot by clicking the mouse button within the plot 4 Scatter plot A scatter plot of data points from the run 5 10 End Point Analysis Calling Allele Types To call allele types Step Action 1 Click the Results tab The software displays the Allelic Discrimination Plot Zoom out until all crossmarks are visible in the plot a b C d D H m BREE EEE Click the amp magnifying glass tool Click the plot to zoom out Click the lasso tool across all datapoints in the plot Select all of the marks within the plot by clicking and dragging the mouse pointer The software outlines all selected wells within the grid view Examine the tray pane to confirm that all wells are selected If not all wells are selected repeat steps a d until all wells are visible on the plot 5 6 7 8 910111213 14 15 16 17 18 19 20 212223 IOUO TEESE RKKK KAAKAA AAAA a IDIKI DIRI I DIDIDE DAIRI DIII DIII IID DIDIDIDIDIPIDIRIDIDIPIDIRIDIDIPIDIRIPIDIPIRIRIE Select the sample cluster exhibiting amplification of the first probe Setup Instrument Results Allele CYP 2C19 2 1 Cal Aleex e el cy cy Legend Allele X Allele Y Allele X amp Y E NTC X Undetermined 2 0 3 0 Allele X CYP 2C19 2 2
218. ox is checked the SDS Software reduces the ramp rate of the 7900HT instrument to match that of the ABI PRiSM 7700 Sequence Detection System instrument 4 14 Run Setup and Basic Operation To create a method for the absolute quantification run continued Step Action 3 Modify the default thermal profile for the method as needed To Then adjust step select a text field value type a new value and press Enter parameters time temp foo Temperature text field between 4 and 99 9 C fi 00 Time text field between 0 01 and 98 59 minutes add a hold a Click step to the left of the location you want to place the cycle set or new stage step b Click Add Cycle or Add Hold The software inserts the stage into the thermal profile Note To adda step to a stage select the step to the left of the location you want to place the step and click Add Step Stage 1 Stage 2 i Stage 3 ea Selected step bso bas New step appears here remove a step a Click the step you want to remove The software highlights the selected step b Click Delete Step to remove the step from the profile 4 Configure the data collection options for the method as follows a Click the Data Collection tab b Click each plateau or ramp within the cycle stage of the thermal profile to place a data collection icon at each step Stage 1 Stage 2 Stage 3 Cycle 0 of 40 i Data collection i
219. p down list Standard Curve Plot Lagend Cor T m Standards Legend box 28 man Bi Unknowns HE T x 5 ann T MENEE Hide Unknowns button 7 mae Oo Standard Curve box a Slope 3 3984165 StH Y Inter 37 091667 3 Z Oe T R2 0 99807143 z THT T ai al Standard curve plot Ha T PTT TT 22 a a e E E H Hl T al H The following table describes the elements of an SDS plate document Component Description Help button Launches the SDS software online help Detector menu Toggles the data displayed within the plot based on detector name Legend box Displays a symbol key for the datapoints appearing in the plot Hide Unknowns button Toggles the presence of data from unknown samples in the plot Standard Curve box Contains statistical data describing the standard curve Item Definition Slope The slope of the standard curve The slope of the standard curve is useful for assessing the efficiency of the assay At 100 efficiency a reaction should achieve a slope of 3 33 since every 10 fold difference in quantity translates to a difference of 3 33 Cys Y Inter The Y axis intercept of the standard curve R2 The R Square value for the standard curve that describes the correlation between threshold cycles C and the log of the starting copy number for the samples that comprise the standard curve plot The calculation yields a value between 1 and 0 where values closer to 1 indicate better cor
220. petting error well lacks template Signal Plateaus Do any of the signals plateau Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal Multicomponent Plot Displays a plot of normalized multicomponent data from a single well of a real time run The plot displays the component dye signals that contribute to the composite signal for the well Correct dyes displayed Does the plot display all dyes as expected The presence of an unexpected dye may be the result of an error in detector setup such as assigning the wrong reporter or quencher dye ROX fluorescence level Does the ROX signal fluoresce below the reporter dyes If not the lack of reporter fluorescence may be caused by an absence of probe in the well a pipetting error Background fluorescence Do all dyes fluoresce above the background The Background signal is a measure of ambient fluorescence If a dye fails to fluoresce above the background it is a strong indication that the well is missing probes labeled with the dye well does not contain probe PCR master mix or both MSE Level The MSE mean squared error is a mathematical representation of how accurately the multicomponented data fits the raw data The higher the MSE value the greater the deviation between the multicomponented data and the raw data Troubleshooting 8 11 8 12 Troubleshooting Trou
221. plate Handler coordinates plate handling for the ABI PRISM 7900HT Sequence Detection System permitting 24 hour unattended operation The arm features a 310 degree rotational swing that permits access to the 7900HT instrument drawer up to five plate stacking areas and the fixed position bar code reader To See Page Turn on the Automation Module 3 5 Align the Automation Module 7 27 Zymark Twister The plate handler consists of the following components Microplate Handler Components 3 E EE Adjustment knob 2 n Plate sensor switch a Gripper cross sectional view of the gripper Plate stack Expansion stacks Zymark Twister Microplate Handler Fixed position bar code reader Product Overview 2 9 Compatible Consumables Consumables for Use with the 7900HT Instrument The ABI PRISM 7900HT Sequence Detection System can support a variety of consumable formats through the use of interchangeable sample block modules Applied Biosystems offers sample block modules that support the following consumable formats Consumable Illustration ABI PRISM 384 Well Reaction Plate 12 13 14 15 16 17 19 20 21 22 23 24 O00O0000000 9 09099 99 GO QO 90 COO OOOOCOO0OCOO6 TOCOOOOOOOOOOCOO SOOOCOOOOOCOOO0 COOCOOCOOCOCOOOOO 0 000900 00000000 SGOOCOCOCOOOOO0O O IO OOOO On OO QO O0OQ
222. plate document 4 6 2a a Create detectors for the absolute quantification probes 4 7 b Copy the detectors to the plate document 4 8 3a a Configure the plate document with detector tasks 4 11 NTC Standard and Unknown b Assign quantities to the wells of the plate document that 4 12 contain standards 4 a Program the method for the absolute quantification run 4 13 b If performing an assay in which you would like to collect 4 16 dissociation data add a temperature ramp to the thermal profile to perform a dissociation curve analysis 5 Choose from the following If running Then a single plate continue to step 7 the first plate in a series Save the plate document as an ABI PRISM of plates with identical SDS Template Document as explained on assay configurations page 4 17 6 Create a plate document from the template created in 4 18 step 5 7 Configure the document with sample names and plate 4 19 information 8 Prepare and run the absolute quantification plate or plates 4 20 9 Analyze the run data 6 3 a Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab delimited text file See Importing Plate Document Setup Table Files on page B 2 for more information Run Setup and Basic Operation 4 3 Allelic To create and set up a plate document for an allelic discrimination run
223. plates 7 18 from a template 4 18 4 32 C See threshold cycle custom pure dyes adding to the pure dye set 7 21 to 7 23 creating a custom pure dye plate 7 21 customer support See technical support cycle set adding toa method 4 15 D DANGER attention word definition 1 2 data collection A 5 data type definitions exportable B 9 decontaminating the sample block module 7 11 to 7 12 defragmenting the hard drive 7 47 deleting steps from a method 4 15 detector tasks about 4 11 applying 4 11 importing into a plate document B 2 detectors about 4 7 applying to a plate document 4 8 copying to aplate document 4 8 creating 4 7 importing into a plate document B 2 tasks See detector tasks determining melting temperatures 6 23 disconnecting the SDS software 4 28 display settings adjusting 4 17 dissociation curve about 6 18 analyzing 6 20 to 6 24 definitions of the T value 6 22 procedure checklist 6 20 programming a temperature ramp 4 16 setting up procedure checklist 4 4 Dissociation Plot about 6 22 exporting as a graphic file B 8 exporting data as atext file B 9 E ejecting a plate from the 7900HT instrument 4 27 4 38 eliminating outliers 6 12 wells from use 6 12 emission filter See instrument optics system end point runs about 5 2 allelic discrimination See allelic discrimination entering bar code information 3 19 4 19 using the Template Batch utility 4 32 comments into a detector 4 7 into a plate doc
224. plates contain can be used to create an unlimited Template File number of plate documents Templates are optional but useful as a time saving devices for experiments where samples are run on plates with identical assay configurations Imported Exported Files Tab delimited txt The SDS software can export raw or analyzed data in text file tab delimited txt format for all or a select group of wells on a plate document The exported files are compatible with most spreadsheet applications JPEGa graphic jpg The SDS software can export most panes and plots of the files plate document as JPEG graphic files The JPEG format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software a Joint Photographic Experts Group The SDS software can produce SDS files of varying sizes depending on the type of runs for which they are created The table below lists the average sizes of typical files produced by the SDS software Run Type Average File Size Compressed File Size Plate read 150 180 KB 70 90 KB Real time 15 25 MB 10 15 MB a Compressed files sizes shown are estimates based on standard compression using the WinZip utility For more information see Archiving SDS Files on page 7 46 b The maximum file sizes displayed above are nominal for real time runs absolute quantification
225. plays the associated information in the well inspector of the Setup tab Move the mouse pointer over After holding position the software displays the any well of the plate grid information for the well in a yellow pop up window Exercise 2 Selecting Multiple Wells The SDS software features several methods for selecting wells from the plate grid The following exercise will familiarize you with most of them To select groups of wells from the plate grid Step Action 1 Select a block of wells from the plate grid by doing one of the following Click and drag the mouse cursor across the block of wells or Click the well at the top left corner of the block then while holding down the Shift key click the well at the bottom right corner of the block The software outlines the selected wells with a black border R Practice sds Ei Practice sds E E e e E Eel e e ABB EE EEEEEE ABBE EEE EEE FEN aad al elle el el eal el el ee Nel leaded eel ell cE BEER EEE ES ic i BEER cae cea ER ERE ES 2 Select several isolated wells of the plate grid by doing of the following a Hold down the Ctrl key and click individual wells to select them The software outlines the selected wells with a black border b While holding down the Ctrl key de select wells by clicking individual selected wells Ej Practice sds TEAS ele ae Elis a i ERE EEEEE amp al al elle Ej Practice sds if2fal4 s elz e a
226. ple Block IMPORTANT Before changing the sample block perform all required upgrades to the SDS software and instrument firmware Failure to upgrade the software can render the instrument inoperable or result in damage to instrument components To replace the sample block Step Action 1 Load the sample block into the instrument compartment as follows a Being careful of the heat sinks on the bottom of the sample block rest the sample block on the metal runners on either side of the instrument bay b Carefully slide the sample block into the instrument until the front of the block is flush with the rear of the locking bar c Once seated firmly press on the sample block to ensure a good connection 2 Replace the sample block locking bar 3 Tighten the thumb screw from step 9 on page 7 6 to secure the sample block locking bar to the instrument chassis may be a 5 32 Hex bolt 4 Using the 5 16 Hex key turn the sample block locking bolt clockwise until it is flush with the locking bar 5 Again press on the right and left sides of the front surface of the sample block to ensure that it is seated securely 6 Replace the thermal cycler access cover as follows a Fit the lip at the bottom of the access cover over the lower edge of the bay b Push the cover towards the instrument until it snaps into place System Maintenance 7 7 7 8 System Maintenance To replace the sam
227. ple block continued Step Action 7 If using a plate handler replace the covers for the fixed position bar code reader and the underlying platform removed in step 6 on page 7 5 I Mia E E E E Fixed position bar code reader and underlying platform covers Plug in and turn on the 7900HT instrument 9 Confirm the function of the installed sample block module as follows a Launch the Automation Controller Software b Click the Thermal Status tab Does the software display temperatures Then Yes the installation is successful The presence of temperature readings confirm that the 7900HT instrument successfully established the connection to the new sample block No the 7900HT instrument is unable to establish communication with the new sample block To troubleshoot the problem a Turn off and unplug the 7900HT instrument b Remove the thermal cycler access cover c Press on the right and left sides of the front plate of the sample block to ensure that it is seated securely d Reinstall the thermal cycler access cover e Repeat step 8 until you hear a high pitched tone confirming communication between the instrument and
228. prise an Authorized Thermal Cycler The purchase price of this Base Unit includes the up_front fee component of a license under United States Patent Nos 4 683 195 4 683 202 and 4 965 188 owned by Roche Molecular Systems Inc and under corresponding claims in patents outside the United States owned by F Hoffmann_La Roche Ltd covering the Polymerase Chain Reaction PCR process to practice the PCR process for internal research and development using this instrument The running royalty component of that license may be purchased from Applied Biosystems or obtained by purchasing Authorized Reagents This instrument is also an Authorized Thermal Cycler for use with applications licenses available from Applied Biosystems Its use with Authorized Reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents Purchase of this product does not itself convey to the purchaser a complete license or right to perform the PCR process Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 DISCLAIMER OF LICENSE No rights for any application including any in vitro diagnostic application are conveyed expressly by implication or by estoppel under any patent or patent applications claiming homogeneous or real_time detection methods including patents covering such methods use
229. pure dye plate from the instrument tray c Extract the pure dye calibration information as explained on page 7 20 System Maintenance 7 19 Extracting Pure Dye The purpose of viewing the data in the Pure Dye Wizard is to eliminate irregular pure Information from dye peaks from the data set The wizard presents the spectral data from the pure dye the Analyzed Run plate in sets of three wells each containing the same pure dye Because the wells displayed by the wizard contain the pure dye at an identical concentration the signal peaks for the set should be identical Occasionally pipetting inaccuracies or contamination can cause a well signal to shift slightly While viewing the data the outlying peaks must be eliminated 7 20 System Maintenance To extract the pure dye information from the run data Step Action 1 From the Analysis menu select Extract Pure Dye Wizard The Extract Pure Dye Wizard dialog appears 2 Follow the instructions as explained by the Extract Pure Dye Wizard to extract the pure dye spectra When presented with each screen do the following a Inspect the spectra for shifts in peak location x Wavelength shift Fe eh eh by Ta re E E E sae A a le eal E a aa el eae Click here to remove it Next Cancel b If the data set contains a outlying peak eliminate it by clicking check box of the associated well Note Dye spectra are generally acceptable if they peak
230. quired to adjust the plate sensor switch Material Part Number ABI PRISM Optical Reaction Plate of the current sample block format See page D 1 The dimensions of different plate formats can place different requirements on how the plate handler grips plates To ensure smooth operation of the automation accessory adjust the plate sensor switch when changing consumable formats To adjust the plate sensor switch Step Action 1 Turn off the Zymark Twister Microplate Handler ite PHYSICAL HAZARD The Zymark Twister Microplate Handler must be turned off at all times during the following procedure Failure to comply can result in physical injury to the user or damage to the plate handler 2 Clear the switch position by turning the thumb wheel all the way to the Up extreme as indicated on the side panel Thumb wheel Plate sensor switch 3 Begin the adjustment of the sensor switch as follows a Grasp a 96 or 384 well ABI PRISM Optical Reaction Plate by the sides making sure not to place pressure in the center of the plate to deform it b Place the plate between the fingers of the gripper assembly and align it to the middle of the centering device c While holding the plate in position slowly turn the thumb wheel to lower the switch onto the reaction plate until the switch Contacts the top of the plate and Emits a soft audible clicking noise IMPORTANT The sound e
231. r or From the File menu select Open 2 From the Look In text field of the Open dialog box navigate to Program Files gt Applied Biosystems gt SDS 2 0 gt Templates 3 From the File of type drop down list select ABI PRism SDS Template Document sdt 4 From the Look In text field click the file entitled 384 Well RNaseP Install Plate sdt to select it 5 Click Open The software opens the plate document file Exercise 4 Exporting Data from a Plate Document In the following exercise you will export the plate setup so that you can import it see Exercise 6 The SDS software allows you to export several components of the plate document as tab delimited text files a format compatible with most spreadsheet applications Note For more information on exporting setup table data using the SDS software see Appendix B Importing and Exporting Plate Document Data To export the contents of the template to a setup table file Step Action 1 Choose one of the following options Click the Export button from the General toolbar or From the File menu select Export From the Export drop down list of the Export dialog box select Setup Table Select the All Wells radio button Click the File name text field and type Practice Click Export O AIOI DN The software saves the plate document setup table information to a tab delimited text file entitled Prac
232. r all or a select group of wells on a plate document The exported files are compatible with most spreadsheet applications and programs that can read tab delimited text To export run data as a tab delimited text file choose one of the following for further instructions See Exporting Plate Document Data on page B 9 Click the help button 1 within the table view Position Sample Na Detector Task Ct Quantity Qty mean Qtystddey 7 Help button Al Ad RNase P Unknown 23 883331 5433 922 A aa PNaca P I Interna 720201413 E762 2A7R O Exporting Plots as Graphics The SDS software can export most panes and plots of the plate document as JPEG Joint Photographic Experts Group graphic files The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software To export a plot as a graphic file see Exporting Graphics on page B 8 or click the help button within the plot of interest for further instructions System Maintenance In This Chapter This chapter discusses the following topics Topic See Page Recommended Maintenance Schedule 7 2 Section Maintaining the 7900HT Instrument 7 3 Replacing the Sample Block 7 4 Changing the 7900HT Plate Adapter 7 9 Decontaminating the Sample Block 7 11 Performing a Background Run 7 1
233. reader and the underlying platform Fixed position bar code reader and underlying platform covers 2 Loosen the three black thumb screws on the platform connecting the 7900HT instrument and the plate handler base KT SeN Black thumb ae screws O 3 Move the instrument tray to the out position a Launch the Automation Controller Software If an error dialog appears reading Machine calibration values are not valid Please refer to documentation for calibration process click OK b Click Open Close Open Close l The 7900HT instrument moves the instrument tray to the out position perpendicular to the instrument c From the File menu select Exit The software quits the Automation Controller Software 4 From the Start menu select Programs gt Zymark Twister Plate Handler gt Twister The Zymark Twister Software launches 5 Click Manual Control The software displays the Manual Control dialog box Aligning The alignment of input stack 1 position 4 in the Zymark Twister Software is the first Input Stack 1 step in the alignment procedure This alignment provides the basis for aligning all Zymark Position 4 Subsequent stacks on the plate handler To align the input stack 1 Zymark position 4 Step Action 1 Place an empty plate into input stack 1 Zymark position 4 2 From the Zymark Twister Software click position 4 Rotary Positioning Click 4 Rotary Adjustment o pam i The pl
234. reen Dye or probes labeled with a non fluorescent quencher The Sequence text value contains the name of the Allelic Discrimination marker associated with the detector Because markers are used exclusively in allelic discrimination plate documents assign Sequence text values to detector definitions of allelic discrimination setup table files only Format for a single detector detector name lt tab gt reporter dye lt tab gt quencher dye lt tab gt description lt tab gt comments lt tab gt sequence lt cr gt Example for an allelic discrimination setup table file CYP ZOIS Zaud PAM PDAR CYP 2C9 2 Allele 1Example ProbeCYP 2C9 2 CYP 2C9 2 2 VIC PDAR CYP 2C9 2 Allele 2Example ProbeCYP 2C9 2 Example for an absolute quantification setup table file GAPDH VIC GAPDH Probe Example Probe SYBR Green SYBR SYBR Green I Example Probe RNase P FAM TAMRA RNase P Probe Example Probe B 6 Importing and Exporting Plate Document Data Setup Table Elements continued Number Contents Description Assay Plate Wells Element numbers 7 and 8 define the contents of the wells on the plate The Assay Plate Wells definition consists of two sections the Well List Header and the Well Definition List 7 Well List This line contains the column headings for the Assay Plate Wells section of the setup table Header file that make the file easier to edit using a program such as Microsoft Excel Format Well lt tab gt
235. relation between C and the log of the starting copy number Note The software calculates the R Square value by taking the square of the Pearson Coefficient of Correlation also known as the rvalue calculated for the data points that comprise the plot The software calculates the R2 value only for the standards that make up the curve Standard curve plot A scatterplot of datapoints from the absolute quantification run 6 14 Real Time Analysis After the Analysis Changing the Plate Document Display Settings Saving the Plate Document Printing a Report Exporting Plate Document Data Before printing or exporting the analyzed data the software allows you to reconfigure the appearance of several elements of the plate document including the results table plate grid and most plots To configure the display settings for the plate document Step Action 1 From the View menu select Display Settings 2 From the Display Settings dialog box click the help button for further instructions on modifying the display settings The software allows you to save any changes made the display settings and or analysis settings baseline and threshold values of the plate document To save the analyzed plate document select Save from the File menu IMPORTANT The Save command saves does not save the results of the analysis only the analysis settings Saved plate documents must be re anal
236. ri M ri M r M ri Ek Es Ee A nr A M ri M ri M ri M ri M ri M ri M ri M ri M nri E E E E E E E E E E E E E E E E E E E E E E_E m Ri M ri M ri M ri M ri M ri M ri M ri M r MA ri Ee eA nR A nr eA nr A ri M ri M ri M nri M ri M ri M ri M ri M ri M ri IE EEEE ERENER EREEREER EEEE ERE EENEN Ri M ri M ri M ri M ri M ri M ri M ri M r MA ri ks eA rR A nr BA nr e ri M ri M ri M pri M ri M ri M pri M ri M ri M ri JE E EEEE Oe he EREEREER EEEREN Ue Ue Ue he Ri M ri M ri M ri M ri M ri M ri M ri M r M ri A nr A rR A nr A A ri M ri M ri M nri M ri M ri M pri M ri Os M nri kE E E E EEEE EREEREER EEEE he Ue Ue Ue Ue ND M ri M ri M ri M ri M ri M ri M ri M ri M ri M r eA r e nr A ri A r E ri M ri M ri M ri M pri M ri M ri M ri M ri M pri TE E E E E E E p E E_E E p E p E E E E E E E EC E Ri M Ri M ri M Ri M ri M ri M ri M ri M ri M or A oR e nr A nri A r E ri M ri M ri M ri M ri M ri M nri M ri M ri M pri TE E E E E E p E E p E E E E E E E E E E E E En M pri M ri M ri M ri M ri M ri M ri M ri M r M nr A rA nr A ri A r E ri M ri M ri M ri M ri M ri M ri M ri M ri M pri MOE E E E E E E E E p E E E E E E E E E E E E EE M pri M ri M ri M ri M ri M ri M r M ri M r es A rA nr A ri A r E ri M ri M ri M ri M ri M ri M ri M ri M ri M pri E E E E E E E E E E E E G G E E E E E E E E ENS m Ri M ri M ri M ri M Ri M Ri M Ri M ri M ri A ri E roi E r E r A nr eA ri M ri M ri M ri M ri M ri M ri M Ri M ri M ri TE E E E E E E E E E E E G GE E E E E E E E E EE Ire aa ik es
237. rm raises to its highest position If the plate contacts the sides of the stack re adjust the rotary position of the plate handler arm until the plate moves freely in the stack Note Contact between the plate and the stack or all stacks may be unavoidable However try to minimize the contact as much as possible To define the positions of the remaining stacks continued Step Action 8 Using the Vertical Positioning commands raise and lower plate handler arm several times to check the alignment 9 Click Rotary Offset and click Yes The software records the rotary position for the Zymark position 5 input stack 2 10 Repeat steps 1 8 for input stacks 3 and 4 to define Rotary Offset values for the remaining positions 6 and 7 Zymark position 7 input stack 4 Zymark position 6 input stack 3 11 Exit the Zymark Twister Software a Click Main Menu The software displays the main menu b Click Exit The software closes 12 Note A bug inthe Zymark Twister Software can cause portions of the program to persist in memory even after the software has been closed Because the Zymark Twister Software conflicts with the SDS software these residual elements must be closed within the Windows Task Manager before continuing Confirm that the Zymark Twister Software has closed by viewing the Task Manager a Press the Ctrl Alt Del keys in unison The Windows Security dialog box appears b C
238. rmal instrument function usually during a run Solid If the light remains on during a Check that the computer is startup for more than 2 min turned on and connected to the The instrument did not boot oe 96 page et kd properly or diagram of instrument connections 7900HT instrument has b If so turn off the instrument wait experienced a system for 30 sec and then restart as failure explained on page 3 5 Red Solid The 7900HT instrument has Turn off the instrument wait for detected a fatal problem 30 sec and then restart Using the SDS Software Workspace Launching the To launch the SDS software either Software Select Start gt Programs gt SDS 2 0 gt SDS 2 0 or Double click the SDS software program icon on the desktop J Accessories j a New Office Document a JE Adobe Ga Open Office Document Norton Antivirus Select SDS 2 0 or Ber F Favorites j A Paint Shop Pro P automation Controller 2 0 3 Documents t Ls LSX MPEG Encoder 3 0 Uninstall SDS 2 0 E Settings r 7C S CS St i it CSt 4 Search j Double click a Help ad Run The computer launches the SDS software and attempts to establish communication with the 7900HT instrument If the connection is successful the software displays the Connected icon Hiv tanmededts Piatename IN the status bar when a plate document is open See About the Status Bar on
239. rop down list select ABI PRism SDS Single Plate sds d Click Save The software saves the plate document The software is now configured for the RNase P run 8 Prepare and run the RNase P plate as explained on page 7 26 System Maintenance 7 25 Preparing and Running an RNase P Plate Verifying Instrument Performance 7 26 System Maintenance To run the RNase P plate Step Action 1 Briefly centrifuge the TaqMan RNase P Instrument Verification Plate 2 From the plate document in the SDS software click the Instrument tab The software displays the Instrument tabbed page 3 From the lower portion of the Instrument tab click the Real Time tab The software displays the Real Time tabbed page 4 If the instrument tray is within the 7900HT instrument click Open Close The instrument tray rotates to the OUT position 5 Place the RNase P plate into the instrument tray Note The A1 position is located in the top left corner of the instrument tray 6 Click Start The 7900HT instrument begins the run Note Before starting the PCR run the instrument may pause up to 15 min to heat the heated cover to the appropriate temperature 7 When the run is complete a Analyze the run data as explained on page 6 9 a Set the baseline and threshold values for the analyzed data as explained on page 6 10 b Verify the performance of the 7900HT instrument as explained below The
240. ropriate section for Allelic Discrimination see page C 5 and for Quantitative PCR see page C 6 Design Probe s for the Assay Adhere to the following guidelines when designing TaqMan probes Keep the G C content in the range of 30 80 Avoid runs of an identical nucleotide especially guanine where runs of four or more Gs should be avoided No Gon 5 end Keep the melting temperature T in the range of 68 70 C for quantitative PCR and 65 67 C for allelic discrimination using the Primer Express software Select the strand that gives the probe with more Cs than Gs For allelic discrimination see page C 5 Adjust probe length so that both probes have the same Tp Position the polymorphism site approximately in the center of each probe For multiplex PCR applications involving multiple probes design the probes with different fluorescent reporter dyes as explained below Reporter Dye Application First Probe Second Probe Allelic Discrimination FAM M VIC a The use of the FAM and VIC reporter dyes for multiplex applications provides the greatest degree of spectral separation Design Primers for the Assay Adhere to the following guidelines when designing primers for 5 nuclease assays Keep the G C content in the range of 30 80 Avoid runs of an identical nucleotide especially guanine where runs of four or more bases should be avoided Keep the T
241. roubleshooting 8 1 Troubleshooting Table Overview The following table is designed to help you troubleshoot most of the problems you may encounter while using the ABI PRISM 7900HT Sequence Detection System The information in the table is arranged by category as follows Chemistry problems Run problems Instrument and Automation Module Problems Each category contains subcategories followed by a brief description of the symptoms you might encounter To use this table look for the category and the symptom you are experiencing The page number in the right hand column corresponds to a description of the possible cause s and recommended action s for that particular problem Table 8 1 Troubleshooting Table Category Symptom Page Chemistry and Run Problems Chemistry Low Precision 8 4 lrreproducibility Run Problems Background Runs Software will not extract background data Background is too high greater than 2500 Pure Dye Runs Software will not extract pure dye data Raw data from pure dye run appears strange Signals plateau saturation Signal is too low lt 10 000 FSU More than two outliers per dye in a single row Real Time Runs Quantitative PCR and Dissociation Curves End Point Runs Allelic Discrimination 8 2 Troubleshooting Table 8 1 Troubleshooting Table continued Category Symptom Page Instrume
242. rs as needed IMPORTANT A marker must be configured with two detectors before it can be applied to a plate document Note Click the 3 button for information on the features of the Marker Manager dialog box or to view the procedure for deleting markers from the Markers list Apply the marker s to the allelic discrimination plate document as explained in Copying and Applying Markers to a Plate Document on page 4 10 Run Setup and Basic Operation 4 9 Copying and IMPORTANT Once copied to the plate document a marker is no longer linked to the Applying Markers to corresponding entry in the Marker Manager Therefore if a marker is modified in the Marker a Plate Document Manager after it has been copied to a plate document the marker must be removed from the plate and copied again to update the plate document with the changes To copy and apply markers to the plate document Step Action 1 From the Marker Manager dialog box of the SDS software copy the allelic discrimination marker to the plate document as follows a Click the marker you want to apply to the plate document The software highlights the selected marker b Click Copy to Plate Document The software copies the marker and associated detectors to the plate document c Repeat steps a and b to copy additional markers to the allelic discrimination plate document as needed Click Done The software closes the Marker Manager dialog
243. run To configure the analysis options for the absolute quantification run Step Action 1 If not already open launch the SDS software as explained on page 4 5 2 Open the plate document file for analysis as follows a From the File menu select Open b From the File Type drop down list select ABI PRism SDS Single Plate sds c From the Look in text field navigate to and select the plate document file d Click Open The SDS software displays the plate document file 3 From the Analysis menu select Analysis Options The Analysis Options Absolute Quantification dialog box appears 4 If desired click the Threshold text field and type an initial threshold value to use for the analysis of the plate document For more information about the threshold setting see Calculating Threshold Cycles on page A 10 Note If no threshold is specified the SDS software will automatically assign a threshold value during the analysis 5 If desired set a default baseline for the analysis a Click the Baseline Start text field and type or dial an initial baseline to use for the analysis of the plate document b Click the Stop text field and type or dial an stop baseline to use for the analysis of the plate document Note If no baseline is specified the SDS software will use the default baseline range of cycles 3 15 for the analysis 6 Set the baseline and threshold values for any remaining detectors present on the plat
244. s N Y pp 303 339 Lee L G Connell C R and Bloch W 1993 Allelic discrimination by nick translation PCR with fluorogenic probes Nucl Acids Res 21 3761 3766 Livak K J Flood S J A Marmaro J and Mullah K B inventors Applied Biosystems Foster City CA assignee 2 Mar 1999 Hybridization assay using self quenching fluorescence probe United States patent 5 876 930 Livak K J Marmaro J and Todd J A 1995 Towards fully automated genome wide polymorphism screening letter Nat Genet 9 341 342 Martens H and Naes T 1989 In Multivariate Calibration John Wiley amp Sons Chichester References E 1 Contacting Technical Support Services and Support Applied Biosystems To access the Applied Biosystems Web site go to Web Site http www appliedbiosystems com At the Applied Biosystems Web site you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Applied Biosystems Web site provides a list of telephone and fax numbers that can be used to contact Technical Support Contacting Technical Support F 1 Limited Warranty Statement Warranty Statement PE Corporation NY through its
245. s and controls the sequence detection run Acquires data during the run Java Runtime Environment It includes additional files and software used to run the SDS software IMPORTANT Do not delete the Java Runtime Environment files These files are crucial to the operation of the SDS software If the files are deleted or become corrupt reinstall the SDS software from the CD as explained on page 7 48 Zymark Twister Software Used to calibrate the Zymark Twister Microplate Handler LAVA Software Used to align the fixed position bar code reader Instrument Firmware Controls the most basic operations of the 7900HT instrument Controlled by commands sent from the computer Acts as the link between the software commands and hardware operations Managing Sequence Detection System Data 7900HT Instrument Data management strategy is a crucial element of successfully integrating the Dataflow ABI PRISM 7900HT Sequence Detection System into a laboratory workflow During a single 24 hour period of real time operation the 7900HT instrument can produce up to 200 MB of data To manage the information produced by the 7900HT instrument successfully it helps to have a basic understanding of how data is collected and processed prior to analysis The figure below contains a summary of the 7900HT instrument data flow ABI PRISM 7900HT Sequence Detection System Instrument Firmware Thermal Cycling an
246. s been run or removed from the queue The ABI PRISM 7900HT Sequence Detection System features several options for adding plate documents to the plate queue Review the options discussed in the following table and choose the method that best suits your needs the Automation Controller Software Option See Page Add a plate document to the plate queue from the SDS software 4 31 Using the Template Batch utility create batches of plate documents from a 4 32 template file and add them to the plate queue Add or remove individual or multiple plate documents to the plate queue using 4 34 4 30 Run Setup and Basic Operation Adding a Plate Document to the Plate Queue from the SDS Software Adding the Plate IMPORTANT A plate document must contain a bar code before it can be added to the plate Document to the queue See page 4 19 for more information on configuring a plate document with bar code Plate Queue information To add the plate document to the plate queue from the SDS software Step Action 1 From the SDS software send the plate document to the queue as follows a Click the Instrument tab The software displays the contents of the Instrument tabbed page b Click the Queue tab The software displays the contents of the Queue tabbed page 2 Click Send to Queue If Then the plate document was the Save As dialog box appears not saved previously a From the Look in text field nav
247. s of the Document Information dialog box or the data it contains click the 7 button to open the contextual online help When finished click OK 4 Run the plate document and associated plate as explained on page 4 20 Run Setup and Basic Operation 4 19 Step 8 Running the Plate on the 7900HT Instrument Options for The 7900HT instrument can run prepared microplates individually or in groups using Running SDS Plates the Zymark Twister Microplate Handler IMPORTANT individually Choose one of the following options to run the plate If you are not using a Zymark Twister Microplate Handler you must run plates Microplate Handler IMPORTANT You must have a Zymark Twister Microplate Handler to run plates using this option Option Description See Page Individual Run the plate individually from the SDS software 4 22 Operation Automated Run the plate with others as part of a batch from the 4 31 Operation Automation Controller Software using the Zymark Twister 4 20 Run Setup and Basic Operation Section Running an Individual Plate In This Section This section discusses the following topics Topic See Page Saving the Plate Document 4 22 Preparing and Running a Single Plate 4 23 Operating the 7900HT Instrument Using the SDS Software 4 25 After the Run 4 27 Run Setup and Basic Operation 4 21 Saving the Plate Document Saving the Plate Before the plate docum
248. s of the experiment can be visualized in the Standard Curve graph of the Results tab The graph consists of a scatter plot of standard and unknown samples graphed on a linear scale plot of Threshold Cycle C versus Starting copy number Real Time Analysis 6 5 Before You Begin Using SDS For specific instructions on any procedure described within this section refer to the Online Help online help accompanying the SDS software To get help at any time during the procedure click a help button located within the dialog box or window in which you are working Examples in This The illustrations and screenshots that appear within this chapter were created for a Chapter TaqMan RNase P Instrument Verification Plate an experiment run during the installation of the 7900HT instrument to verify its performance The sealed plate is pre loaded with the reagents necessary for the detection and quantification of genomic copies of the human RNase P gene a single copy gene encoding the moiety of the RNase P enzyme Each well contains pre loaded reaction mix 1X TaqMan Universal PCR Master Mix RNase P primers and FAM labeled probe and template The following figure illustrates the arrangement of standards and samples on the RNase P plate As shown below the RNase P plate consists of 5 columns of template standards 1250 2500 5000 10 000 and 20 000 copies and two unknown populations 5000 and 10 000 copies 15 16 17 18 19 20 2324 A
249. s part of the calibration file located in the Calibration subdirectory of the SDS 2 0 directory To extract the background component from the run data Step Action 1 From the Analysis menu select Extract Background The software attempts to extract the background signal and displays the success of the extraction in a dialog box Dialog Box Then xi the run is successful Background calibration information updated The raw spectra read from the Background plate conform to acceptable limits Proceed to step 3 5l the run is unsuccessful xl The signal measured in some ofthe wells is too high and indicates block contamination x This data is not usable for background extraction The software has stopped the extraction because one or more raw spectra exceed 2500 FSU Troubleshoot and decontaminate the sample block as explained in Background Runs on page 8 8 2 From the File menu select Save The software saves the plate document 3 From the File menu select Close The software closes the plate document 7 16 System Maintenance Performing a Pure Dye Run When to Perform Purpose of Pure Dye Runs Components of the Pure Dye Spectra Applied Biosystems recommends performing spectral calibration Every 6 months depending on instrument use After changing sample block formats see page 7 4 IMPORTANT Always run a background plate bef
250. sample block 10 Once the sample block is loaded into the instrument do the following a Perform a background run see page 7 13 to verify that the sample block Is connected and working properly Contains no contaminants that will interfere with fluorescent detection b If changing sample block formats perform any remaining tasks outlined in the Sample Block Installation Checklist on page 7 4 Changing the 7900HT Plate Adapter When to Perform Remove and replace the 7900HT instrument plate adapter after changing the sample block module format for example replacing a 384 well sample block module with a 96 well version Note The sample block must be used with the corresponding plate adapter of the same plate format Materials Required The procedure below requires the use of the following materials Material Part Number One of the following 384 Well Plate Adapter See page D 3 96 Well Plate Adapter 3 32 inch Hex key Changing the Plate To replace the 7900HT instrument plate adapter Adapter Step Action 1 If the instrument tray is inside the 7900HT instrument move the instrument tray to the OUT position as follows a Launch the SDS software b From the File menu select New The New Document dialog box appears c Click OK The software generates a plate document d Click the Instrument tab e From the Real Time tab of the Instrument tabbed page click
251. set orstep 4 15 temperature ramp 4 16 adjusting step parameters 4 15 configuring data collection options 4 15 programming 4 14 to 4 16 removingastep 4 15 setting the sample volume 4 15 multicomponenting A 5 N no template control NTC detector task applying 4 11 NTC calls applying verifying normalization of reporter signals A 6 normalized reporter signal A 6 Note attention word definition 1 2 O Opening instrument tray from the Automation Controller software 4 38 from the SDS software 4 27 plate documents 3 13 operating system Supported 2 7 upgrading 7 47 operating the 7900HT instrument power switch 3 5 optimizing primer and probe concentrations C 4 outliers absolute quantification 6 12 allelic discrimination 5 6 P panes hiding 3 16 maximizing minimizing 3 15 resizing 3 15 showing 3 16 passive reference setting 4 12 use in multicomponent analysis A 5 PCR 5 Nuclease Assay A 2 Geometric Exponential Phase A 7 kinetic analysis of A 7 Linear Phase A 8 Plateau Phase A 8 SYBR Green Chemistry A 3 pipetting errors 8 5 pipettors using 8 5 plate adapter changing 7 9 plate document information applying to a plate document 4 19 plate documents 4 6 about 3 11 adding to the plate queue 4 31 4 35 applying detector tasks 4 11 applying sample names 4 19 assigning standard quantities 4 12 closing 3 14 configuring document information 4 19 copying detectors 4 8 copying markers 4 10 creating 3 12 4 6 from a templat
252. sit the Applied Biosystems Online Store http store appliedbiosystems com or Email Applied Biosystems with your order OligosUS appliedbiosystems com Part Number Description TaqMan MGB Probes 5 Fluorescent label 6 FAM VIC or TET 4316034 5 000 6 000 pmols 4316033 15 000 25 000 pmols 4316032 50 000 100 000 pmols TaqMan Probes 5 Fluorescent label 6 FAM VIC or TET 3 label TAMRA 450025 5 000 6 000 pmols 450024 15 000 25 000 pmols 450003 50 000 100 000 pmols Sequence Detection Primers 4304970 Minimum 4 000 pmols purified for sequence detection 4304971 Minimum 40 000 pmols purified for sequence detection 4304972 Minimum 130 000 pmols purified for sequence detection Kits Reagents and Consumables D 5 References Bloch W 1991 A biochemical perspective of the polymerase chain reaction Biochemistry 30 2735 2747 Forster V Th 1948 Zwischenmolekulare Energie wanderung und Fluoreszenz Annals of Physics Leipzig 2 55 75 Higuchi R Dollinger G Walsh P S and Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences BioTechnology 10 413 417 Higuchi R Fockler C Dollinger G and Watson R 1993 Kinetic PCR analysis Real time monitoring of DNA amplification reactions Bio Technology 11 1026 1030 Lakowicz J R 1983 Chapter 10 Energy Transfer In Principles of Fluorescent Spectroscopy Plenum Pres
253. sociation Curve Analysis2 a For dissociation curve analysis the instrument collects data during a pre programmed temperature ramp and plots the negative of the first derivative for normalized fluorescence over time Note For detailed information on any of the chemistries supported by the ABI PRISM 7900HT Sequence Detection System see the appropriate chapter for the type of run see Chapter 5 End Point Analysis and Chapter 6 Real Time Analysis respectively Section Getting to Know the Hardware In This Section This section contains the following information Topic See Page 7900HT Instrument 2 4 Computer 2 7 Bar Code Readers 2 8 Zymark Twister Microplate Handler 2 9 Compatible Consumables 2 10 Instrument Connections 2 11 Instrument The ABI PRISM 7900HT Sequence Detection System consists of the following Components components Number For information on the See Page 1 7900HT Instrument 2 4 2 Fixed Position Bar Code Reader 2 8 3 o Accessory Zymark Twister Microplate Handler a 4 Extended Capacity Stacks 5 Hand Held Bar Code Reader 2 8 6 Microsoft Windows Compatible Computer 2 7 Product Overview 2 3 7900HT Instrument Internal The 7900HT instrument contains the hardware used for thermal cycling
254. software To export an element of a plate document as a graphic Step Action 1 Click the plot or grid you want to export 2 Choose from the following If exporting a plot adjust its dimensions length and width as you want them to appear in the exported graphic file The exported graphic file retains the dimensions of the original screen element If exporting the plate grid do not adjust the size of the wells The software captures the whole grid regardless of the size of the view Right click the plot or grid and select Save Plot Grid to Image File from the contextual menu Note Ifa pane cannot be exported as a graphic the contextual menu will not contain the Save Plot Grid to Image File option From the Save As dialog box navigate to the directory you want to receive the exported graphic file Click the File name text field and type a name for the new file Click Save The software saves the plot or grid as a JPEG graphic in the designated directory B 8 Importing and Exporting Plate Document Data Exporting Plate Document Data Exporting Data from a Plate Document The SDS software can export raw or analyzed data in tab delimited txt format for all or a select group of wells on a plate document The exported files are compatible with most spreadsheet applications To export an element of a plate document Step Action 1 Click the plate document t
255. sor switch is contacting the plate The gripper does not contact the side of the stack 9 Click Close Gripper The gripper grips the plate between its fingers 10 Select Vertical Home The plate handler raises the arm to its highest position If the plate contacts the sides of the stack re adjust the rotary position of the plate handler arm until the plate moves freely in the stack Note Contact between the plate and the stack or all stacks may be unavoidable However try to minimize the contact as much as possible 11 Using the Vertical Positioning commands raise and lower plate handler arm several times to check the alignment 12 Lower the plate handler arm and click Rotary Offset and click Yes The software records the rotary position for position 0 the output stack System Maintenance 7 37 To find the bottom of the stack continued Step Action 13 Click the Vertical Home 14 While holding the plate click Open Gripper and remove the plate 15 Click the Vertical Adjustment text field type 3200 and press Enter The plate handler lowers the arm to a position near the base of the output stack 16 Carefully lower the plate handler arm until it is approximately 1 2 mm from the bottom of the stack 17 Click Vertical Offset click Yes and record the number in the Vertical Adjustment text field The software records the vertical position for position O the output s
256. special or consequential loss damage or expense directly or indirectly arising from the purchase or use of the Instrument Applied Biosystems makes no warranty whatsoever with regard to products or parts furnished by third parties This Warranty is limited to the original location of installation and is not transferable THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS G 2 Limited Warranty Statement Index Symbols gt symbol 3 4 Numerics 7900HT instrument See instrument 9600 emulation mode 4 14 A ABI Prism 7700 Sequence Detection System emulating 4 14 ABI PRISM SDS Single Plate sds files See plate documents ABI PRISM SDS Template Document sdt files See templates absolute quantification about 6 4 to 6 5 analyzing data 6 7 to 6 14 assay development guidelines C 6 procedure checklist 6 7 selecting and preparing standards C 6 setting up procedure checklist 4 3 troubleshooting 8 11 adding bar code to a plate document 3 19 4 19 custom dyes to the pure dye set 7 21 detector tasks to a plate document 4 11 detectors to a plate document 4 8 markers to a plate document 4 10 plate documents to the plate queue 4 31 4 35 sample names to a plate document 4 19 adjusti
257. stablished in the GeneAmp PCR System 9700 Modules and thermal cycler Accessories The use of an interchangeable sample block module Reduces instrument downtime by allowing immediate replacement of the block Permits easy access to the sample block for troubleshooting and maintenance see page 7 11 Supports multiple consumable formats Provides several different modes of operation including Max mode and programmable temperature ramps Part No Description Quantity a 384 Well Interchangeable Sample Block Module for the ABI PRISM 7900HT Sequence Detection System Includes a 384 Well Sample Block Module a 384 well plate adapter and a Sequence Detection Systems 384 Well Spectral Calibration Kit P N 4323977 1 kit 96 Well Interchangeable Sample Block Module for the ABI PRISM 7900HT Sequence Detection System Includes a 96 Well Sample Block Module a 96 well plate adapter and an ABI PRISM 7900HT Sequence Detection Systems 96 Well Spectral Calibration Kit P N 4328639 1 kit a contact your local Applied Biosystems Sales and Service Office for information Kits Reagents and Consumables Disposables 4 Consumables and The ABI PRISM 7900HT Sequence Detection System can run both ABI PRISM Optical 384 Well Reaction Plates sealed with ABI PRISM Optical Adhesive Covers ABI PRISM 96 Well Reaction Plates sealed with ABI PRISM Optical Adhesive Covers or ABI P
258. strument tray must be empty to begin a run If the instrument tray contains a plate eject and remove it before continuing The output stack does not contain plates Plate Requirements See Consumables and Disposables on page D 3 for a complete list of ABI PRISM consumables and ordering instructions Guidelines Observe the following guidelines when loading plates onto the plate handler Before loading plates onto the plate handler make sure that for each plate the associated plate document has been added to the plate queue the plate has been sealed using an optical adhesive cover Load the plates into the plate handler stacks in any order The software reads the bar code of each plate before it is run and matches the plate document and method with the plate The Automation Controller Software can run batches of up to 84 plates in a single session 21 plates stack Orient the plates within the stacks so that well A1 e of each plate corresponds to the locations shown in the illustration below Do not place plates in the output stack X The Zymark arm uses the empty stack to store used plates after they are run Bar code Zymark Twister Microplate Handler S wen A1 top view 4 36 Run Setup and Basic Operation Loading Plates To load plates onto the automation module Step Action 1 Following the guidelines on page 4 36 load the sealed plates in
259. t of ABI PRISM consumables and ordering instructions Preparing the Plate To prepare the plate Step Action 1 Prepare the reactions in an ABI PRISM Optical Reaction Plate by aliquoting reagents enzyme and samples to the appropriate wells of an optical plate IMPORTANT The arrangement of the reactions samples and assays on the plate must match the configuration of the corresponding plate document Seal the ABI PRISM Optical Reaction Plate with an ABI PRISM Optical Adhesive Cover or ABI PRISM Optical Flat Cap Strips Briefly centrifuge the plate to collect the reactions at the bottom of the wells and to eliminate any air bubbles that may be present Choose from the following Experiment Then Absolute Quantification Run the plate as explained in Running the Dissociation Curve Analysis Plate on page 4 24 Allelic Discrimination a Load the plate onto a designated thermal cycler and perform the PCR b Briefly centrifuge the plate to draw the reactions to the bottom of the wells and to eliminate any air bubbles that may have formed during thermal cycling c Run the plate as explained in Running the Plate on page 4 24 Run Setup and Basic Operation 4 23 Running the Plate To begin the single plate run Step Action 1 From the SDS software click the Instrument tab of the plate document The software displays the Instrument tabbed p
260. tack 18 Click Vertical Home The plate handler raises the plate handler arm to its highest position 19 Click the Vertical Adjustment text field type the Vertical Offset value determined in step 5 and press Enter The plate handler lowers the plate handler arm to a Vertical Offset position 20 If necessary readjust the Vertical Offset value and repeat steps 6 and 7 until satisfied with the setting Defining the To define the positions of the remaining stacks Positions of the Remaining Stacks 7 38 System Maintenance Step Action 1 Place an empty plate into input stack 2 Zymark position 5 2 From the Zymark Twister Software click position 5 The plate handler arm moves over the input stack Using the Vertical Positioning commands lower the plate handler arm until it is approximately 1 cm above the stack and center it using the Rotary Adjustment arrows Carefully lower the plate handler arm into the stack Center the gripper as it moves down the stack by adjusting the Rotary Adjustment arrows as needed Once the plate handler arm is centered within the stack click Find Plate The plate handler arm lowers upon the plate Confirm the following The plate is in the middle of the gripper span The plate sensor switch is contacting the plate The gripper does not contact the side of the stack Click Close Gripper Click Vertical Home The plate handler a
261. tatus of software functions and a instrument status icon for indicating the status of the instrument About the Message Bar The message bar displays a variety of messages to indicate the status of the instrument The following table summarizes all of the messages displayed in the Message bar Message Then the SDS software is Ready ready and awaiting instructions Collecting Data currently running a plate document Reanalyze data requesting analysis of plate document data The Analysis Options for the plate document have been changed and the document requires reanalysis for them to take effect Analyzing data Progress bar completing the calculations for the current analysis The metered bar to the right of the message displays the progress of the analysis Saving data Progress bar saving the plate document or template to a storage device The metered bar to the right of the message displays the progress of the action Importing data importing a file The metered bar to the right of the message displays the progress of the action Exporting data Progress bar exporting the data within the current plate document to a file The metered bar to the right of the message displays the progress of the action About the Instrument Status Icon Indicates the status of the connection to the 7900HT instrument Icon Instrument Status Ely Connected to PlateNa
262. te Batch Utility About the Template The Template Batch utility allows you to quickly create multiple plate documents from Batch Utility a single ABI PRISM SDS Template file sdt The Template Batch utility can be a useful time saving device in situations where samples are run on plates with identical assay configurations IMPORTANT Plate documents created by the Template Batch utility do not contain sample or plate information This information must be applied to each plate document individually after the file is run Generating Plate Note For more information on the elements of the Template Batch dialog box or to view the Documents From a Procedures for importing or editing Plate IDs click the _ button and see the SDS software Template online help To create plate documents from a template using the Template Batch Utility Step Action 1 From the SDS software open a template file as follows a From the File menu select Open b From the File Type drop down list select ABI PRISM SDS Template Document sdt c From the Look in text field navigate to and select the template file d Click Open The SDS software displays the template file 2 Send the plate document to the queue as follows a Click the Instrument tab The software displays the contents of the Instrument tabbed page b Click the Queue tab The software displays the contents of the Queue tabbed page 3 Click Send to Queue The Template
263. ted Check the seal of the optical adhesive cover for leaks End Point Runs Allelic Discrimination Troubleshooting When faced with irregular data you can use the SDS software to diagnose some Analyzed Data from chemistry and instrument related problems The following table contains a summary an End Point Run Of checks for verifying the integrity of your run data and to help you begin troubleshooting potential problems Troubleshooting Analyzed End Point Run Data Analysis View Description What to look for Raw Data Signal tightness and uniformity Do the raw spectra signals from replicate groups and controls exhibit similar spectral profiles If not the plate or sample block could be contaminated Displays the raw reporter fluorescence signal not normalized for the selected wells during each cycle of Characteristic signal shape Do the samples peak at the the PCR expected wavelengths For example samples containing only FAM labeled TaqMan probes should not produce raw fluorescence in the peak wavelength of the VIC dye component A signal present in wells that do not contain the dye could indicate that the sample master mix or well contains contaminants Signal Plateaus Do any of the signals plateau Signal plateaus or saturation can be an indication that a well contains too much template or fluorescent signal Troubleshooting 8 13 Software and 7900HT Instrument Troub
264. tep is to align the plate handler arm to the instrument tray Zymark Handler to the position 2 This alignment will ensure a smooth exchange between the plate handler Instrument arm and the instrument tray during operation of the instrument Note The following procedure requires you to position the plate handler relative to the 7900HT instrument Before moving the plate handler loosen the three black thumb screws on the platform connecting the 7900HT instrument and the plate handler To adjust the plate handler relative to the 7900HT instrument Step Action 1 If not already present place an empty plate into input stack 1 Zymark position 4 and pick it up with the plate handler arm as follows a From the Zymark Twister Software click position 4 b Click Find Plate c Click Close Gripper 2 Click position 2 Click here The plate handler arm moves over the instrument tray 3 Use Vertical Positioning to lower the plate handler arm until it is approximately 1 cm above the instrument tray 4 Using the Rotary Adjustment arrows center the gripper and plate along the Y axis of the instrument tray N Y Y Center the plate K A 5 Center the gripper and plate along the X axis of the instrument tray by sliding the plate handler and base towards or away from the 7900HT instrument
265. tes on the 7900HT instrument 9 Analyze the run data 5 9 10 View the results of the allelic discrimination run 5 10 11 Call allele types for each marker 5 11 12 Scrutinize the allele calls 5 13 13 Choose from the following post analysis options 5 15 Reanalyze the run data Adjust the display settings for the plate document Print elements of the plate document Export the plate document results table or plots a Steps 2 and 3 can be eliminated by importing the plate document setup information from a tab delimited text file See Importing Plate Document Setup Table Files on page B 2 for more information 5 8 End Point Analysis Analyzing a Completed Allelic Discrimination Run Analyzing the Run To analyze a completed allelic discrimination run Step Action 1 If not already open launch the SDS software as explained on page 4 5 2 Open the plate document file for analysis as follows a From the File menu select Open b From the Look in text field navigate to and select the plate document file c Click Open The SDS software displays the plate document file 3 Choose one of the following From the Analysis menu select Analyze From the toolbar click the Analyze button B The SDS software analyzes the run data and displays the results in the Results tab End Point Analysis 5 9 Calling and Scrutinizing
266. th no template control or genomic DNA If no good sequence is found it may be necessary to examine the sequence and redesign the amplicon or simply screen for more sites If the gene you are studying does not have introns then you cannot design an amplicon that will amplify the mRNA sequence without amplifying the genomic sequence In this case it may be necessary to run RT minus controls Selecting and To ensure accurate results the standards used for absolute quantification must be Preparing Standards carefully engineered validated and quantified before use Consider the following for Absolute Critical points for the proper use of absolute standard curves Quantification C 6 Designing TaqMan Assays The DNA or RNA used must be a single pure species For example plasmid DNA prepared from E coli often is contaminated with RNA which increases the Apso measurement and inflates the copy number determined for the plasmid In general DNA cannot be used as a standard for absolute quantification of RNA because there is no control for the efficiency of the reverse transcription step Absolute quantities of the standard must be known by some independent means Plasmid DNA or in vitro transcribed RNA are commonly used to prepare absolute standards Concentration is measured by Azg and converted to the number of copies using the molecular weight of the DNA or RNA Consider the stability of the diluted standards especially for RNA D
267. the setup table file 2 Import the setup table file from the previous procedure as tab delimited text If using a spreadsheet application to edit the setup table file the application automatically parses the tab delimited information into the cells of a soreadsheet 3 Configure the setup table file with sample and detector information according to the file structure explained on page B 5 Save the setup table file in tab delimited format Import the completed setup table file into an empty plate document as explained below Importing the Completed Setup Table File into a Plate Document The final step in the procedure is to import the completed setup table tab delimited file into an empty plate document To import setup information from a tab delimited text file to a plate document Step Action 1 If the plate document created in Creating an Empty Setup Table File on page B 2 is still open in the SDS software continue to step 3 Otherwise create a plate document to receive the setup table data as follows a Launch the SDS software b Create or open a plate document to receive the information from the text file 2 Choose one of the following options From the toolbar click the Import button E From the File menu select Import 3 From the Look In text field of the Import dialog box navigate to and select the completed tab delimited setup table file from step 4 in the previous proc
268. tice txt Note The software also can export the data from most of the analysis plots graphs and tables See Appendix B Importing and Exporting Plate Document Data for more information Getting Started 3 13 3 14 Getting Started Exercise 5 Closing a Plate Document When finished viewing or editing a plate document you will need to close it If the plate document has been altered since last saving it the software will prompt you to save the document In the following procedure you will close the template document opened in Exercise 3 To close a plate document Step Action 1 From the File menu select Close 2 If prompted to save the plate document click No The SDS software closes the file without saving it Exercise 6 Importing Setup Table Data into a Plate Document As a time saving device the SDS software allows you to import a setup table information into a plate document from an exported tab delimited text file To illustrate this feature import the plate grid setup information contained in the Practice txt file from Exercise 4 into the empty plate document created in Exercise 1 To import a setup table file into an empty plate document Step Action 1 If the plate document from Exercise 1 is still open go to step 2 Otherwise create a new plate document to receive the setup table data as follows a From the File menu select New b Configure the New Docum
269. tion Rerun the pure dye plate If the problem persists discard the pure dye plate and run a new one Real Time Runs Quantitative PCR and Dissociation Curves Troubleshooting When faced with irregular data you can use the SDS software to diagnose some Analyzed Data from chemistry and instrument related problems The following table contains a summary a Real Time Run Of checks for verifying the integrity of your run data and to help you begin troubleshooting potential problems Troubleshooting Analyzed Real Time Run Data Analysis View Description What to look for Raw Data Plot Displays the raw reporter fluorescence signal not normalized for the selected wells during each cycle of the real time PCR Signal tightness and uniformity Do the raw spectra signals from replicate groups and controls exhibit similar spectral profiles If not the plate or sample block could be contaminated Characteristic signal shape Do the samples peak at the expected wavelengths For example samples containing only FAM labeled TaqgMan probes should not produce raw fluorescence in the wavelength of a VIC dye component A signal present in wells that do not contain the dye could indicate that the sample master mix or well contains contaminants Characteristic signal growth As you drag the bar through the PCR cycles do you observe growth as expected Absent growth curves may indicate a pi
270. tion Plates Fluorescent Contamination on the Plates Errors 8 6 Troubleshooting Drops of reagents that cling to the sides of the wells may not contact the thermal cycler sample block and consequently may not amplify If the drop slides into the mix during PCR then the amplified products will become diluted and the final result will be less than replicate wells that did not have drops Therefore carefully monitor the reaction plate as it is being transferred into the thermal cycler or 7900HT instrument If you observe any drops take steps to remove them such as centrifugation Do not write on any surface of the Optical 384 96 Well Reaction Plates or the Optical Adhesive Covers The fluorescent properties of the ink can potentially affect the fluorescence emission from the plate and alter the results Instead note the contents of each well on a sheet of paper or on a printout of the sample setup Many compounds found in laboratories are fluorescent If they come in contact with certain optical surfaces such as the optical adhesive covers the fluorescent results may be affected For example it has been noted that the powder used to lubricate the insides of plastic gloves often contains fluorescent compounds Use only powder free gloves and do not needlessly touch the reaction plates or optical adhesive seals Human errors from time to time are inevitable such as pipetting into the wrong well or making a dilution mistake Human err
271. to the plate handler stacks 2 From the Automation Controller Software select the check boxes for the plate stacks containing plates IMPORTANT Ifyou are not using stack 1 or the Restack option explained below remove all plates from stack 1 before starting the queue Under these settings the plate handler will attempt to stack the run plates from stack 2 in the stack 1 position If stack 1 contains plates these settings will cause the plate handler to stop the run Instrument Control Robot s Active Stacks Open Close a Stack 7 Stack2 7 Stack3 M cath Restack when finished Plate stack check Online boxes 3 If you want to retain the location of the plates in the stacks on the plate handler select the Restack when finished check box When selected the Restack function instructs the arm to replace a stack of used plates to their original stack and in their original order after the stack has been run If the option is not selected the arm will place each group of used plates within the next vacant stack in clockwise order beginning with the Output stack Note Restacking plates adds significant operating time when running multiple plates Use the Restack function only when absolutely necessary 4 Begin the plate queue as explained on page 4 38 Run Setup and Basic Operation 4 37 Operating the 7900HT Instrument Using the Automation Controller Software Starting the Plate Queue Monitoring
272. tom Dyes to the Pure Dye Set 7 21 Verifying Instrument Performance Using a TaqMan RNase P Plate 7 24 System Maintenance 7 3 Replacing the Sample Block When to Perform Sample Block Installation Checklist IMPORTANT Before changing the sample block perform all required upgrades to the SDS software and instrument firmware Failure to update the software can render the instrument inoperable or result in damage to instrument components You will need to remove the 7900HT instrument sample block when Decontaminating the wells of the sample block see page 7 11 Changing sample block formats IMPORTANT Always run a background plate after installing the sample block Unless instructed to do otherwise adhere to the following guidelines when exchanging sample block modules of different formats To install a sample block module Step Procedure See Page 1 Perform all required software and firmware upgrades to the ABI PRISM 7 48 7900HT Sequence Detection System 2 Remove the existing sample block 7 5 3 Install the new sample block 7 7 4 Change the plate adapter 7 9 5 Run a background plate to check the sample block for contamination 7 13 If changing block formats or if installing a new block also perform the following 6 Run a pure dye plate to create the pure spectra calibration values for 7 17 the new format 7 Run an TaqMan RNase P Instrument Verification Plat
273. tom pure dye plate is identical to that of an ABI PRISM Pure Dye Plate The SDS software uses the custom plate to create a spectral standard for multicomponenting the custom dye To create a pure dye plate for custom dyes Step Action 1 Prepare a microplate with a dilution series of the custom dye 2 Launch the SDS software 3 Create an allelic discrimination plate document and run the dilution series plate Note Itis not necessary to configure detector sample and method information for the dilution series plate document The purpose of the run is to establish the correct working concentration for the dye by viewing the intensity of the raw spectra produced by the wells in the dilution series 4 From the Analysis menu select Analyze The software analyzes the raw run data 5 Click the Show Raw Data Plot button fj from the Display toolbar The software displays the Raw Data Plot 6 From the raw spectra determine the highest concentration of dye that does not produce a saturated signal and record it for future use Note Saturated signals are characterized by their high peaks that rise beyond detectable levels gt 65 000 fluorescent units and appear as plateaus on the Raw Data plot The concentration of the custom dye that yields the highest possible signal but does not saturate is the maximum concentration for use with the 7900HT instrument 7 Repeat steps 1 to 6 for any additional custom dyes
274. topping a run during thermal cycling can affect the chemistry of the reactions within the plate Before stopping a run carefully read the guidelines on page 4 26 to determine the best course of action To eject a plate following a halted run click Open Close from the Automation Controller Software window The run can be analyzed immediately following the completion of the run To analyze data from a plate containing assays for See Page Allelic Discrimination 5 3 Absolute Quantification 6 3 Dissociation Curve Analysis 6 17 4 38 Run Setup and Basic Operation End Point Analysis In This Chapter This chapter discusses the following topics Topic See Page End Point Runs on the 7900HT Instrument 5 2 Section Allelic Discrimination 5 3 Overview 5 4 Before You Begin 5 7 Analysis Checklist 5 8 Analyzing a Completed Allelic Discrimination Run 5 9 Calling and Scrutinizing Allelic Discrimination Data 5 10 After the Analysis 5 15 End Point Analysis 5 1 End Point Runs on the 7900HT Instrument End Point Runs _ End point is the term used to describe the category of sequence detection runs in 5 2 End Point Analysis which the ABI PRISM 7900HT Sequence Detection System is used to measure the fluorescence of a biological sample after it has undergone thermal cycling Unlike real time runs that can yield quantitative measuremenis the focus of end point experi
275. ts are virtual representations of the consumables MicroAmp 384 96 well plates used to contain samples and reagents during runs Plate documents contain the following information Detector information and arrangement on the plate Marker information and arrangement on the plate allelic discrimination only Sample information and their arrangement on the plate Method parameters for the run absolute quantification only Creating a Plate To create a plate document Document Step Action 1 If not already open launch the SDS software as explained on page 4 5 2 Choose from the following From the File menu select New or From the tool bar click the New Document button O 3 Configure the New Document dialog box with settings for the run as follows Drop Down List Select Assay the type of assay appropriate for your plate Note If performing a dissociation curve experiment select Absolute Quantification from the Assay drop down list Container the type of plate you intend to run Template Blank Template x fa Assay drop down list Assay Absolute Quantification Container 384 Wells Clear Plate Container drop down list Template drop down list Template Blank Template Browse Barcode arce Barcode text field Leave blank Note Because this chapter emphasizes the use of a template file the Barcode text field is left blank
276. uence Detection System is used to measure the fluorescence of a biological sample during thermal cycling In contrast to end point runs real time experiments can be used to achieve both qualitative and quantitative measurements Real time analysis can be used in combination with either TagMan or SYBR Green 1 double stranded DNA binding dye chemistry for a variety of purposes including quantitative PCR and dissociation curve analysis Quantitative RT PCR is a method used to measure small quantities of ribonucleic acid sequences isolated from biological samples Typical biological samples include cells tissues and fluids During the RT step reverse transcription of target RNA produces corresponding complementary DNA cDNA sequences During the subsequent PCR the initial concentration of target cDNA is quantified by amplifying it to a detectable level There are two types of quantitative RT PCR Absolute quantification Relative quantification Section Absolute Quantification In This Section This section contains the following information Topic See Page Overview 6 4 Before You Begin 6 6 Analysis Checklist 6 7 Analyzing the Run Data 6 8 Viewing Results 6 13 After the Analysis 6 15 Real Time Analysis 6 3 Overview About Absolute Quantification Employing the 5 Nuclease Assay 6 4 Real Time Analysis The ABI PRISM 7900HT Sequence Detection System supports real time absolute quanti
277. uences present in the sample A substantial increase in Indicates VIC fluorescence only homozygosity for Allele X FAM fluorescence only homozygosity for Allele Y both fluorescent signals heterozygosity 5 4 End Point Analysis Algorithmic Manipulation of Raw Allelic Discrimination Data Cluster Variations The SDS software can analyze raw data immediately upon completion of an allelic discrimination run The term raw data refers to the spectral data between 500 nm to 660 nm collected by the SDS software during the plate read During the analysis the software employs several mathematical algorithms to generate from the raw data a more direct measure of the relationship between the spectra changes in the unknown samples The first mathematical algorithm involves the conversion of the raw data expressed in terms of Fluorescent Signal vs Wavelength to pure dye components using the extracted pure dye standards After the dye components have been identified the software determines the contribution of each dye in the raw data using the multicomponent algorithm See Multicomponenting on page A 5 for a complete description of the process The SDS software graphs the results of an allelic discrimination run on a scatter plot contrasting reporter dye fluorescence Allele X R versus Allele Y R The software represents each well of the 384 well plate as a datapoint on the plot The clustering
278. ument 4 19 sample names into a plate document 4 19 exportable data type definitions B 9 exporting data from a plate document 3 13 B 9 plots and views as graphic files B 8 F finger pads cleaning 7 43 replacing 7 43 fixed position bar code reader aligning 7 40 to 7 42 connections 2 11 location 2 8 specification 2 8 fluorescent contamination 8 6 detection system 2 11 A 4 fluorogenic probe about A 2 designing C 2 to C 3 G genotypic segregation of datapoints Allelic Discrimination Plot 5 6 Geometric Exponential Phase A 7 grating See instrument optics system grey dividing line using 3 15 grid See plate grid gripper 7 27 guidelines assay development absolute quantification C 6 allelic discrimination C 5 loading the plate handler 4 36 setting the baseline 6 10 setting the threshold 6 11 TaqMan probe design C 2 to C 3 H hand held bar code reader connections 2 11 location 2 8 specification 2 8 using 3 19 hard drive defragmenting 7 47 partitions 2 7 heated clamp 2 4 help background information 3 3 using the SDS Online Help 3 3 hold adding to a method 4 15 hotkey combinations using 3 20 I Important attention word definition 1 2 importing setup table data 3 14 B 2 imprecise pipetting 8 5 improper threshold setting 8 4 Index 3 I continued installing plate adapter 7 9 sample block module 7 4 SDS software 7 48 the operating system software 7 47 instrument 2 4 about 2 2 to 2 11 external compone
279. urn the thumb wheel in the Down direction 96 Well 20 steps 384 Well 15 steps Note If you lose count begin again from step 4 and identify the zero point for the switch 6 Test the adjustment as explained on page 7 30 System Maintenance 7 29 Testing the To test the sensitivity of the plate sensor switch Adjustment Step Action 1 Place the reaction plate in the input stack 1 of the plate handler 2 Turn on the 7900HT instrument the plate handler and the computer 3 From the Start menu select Programs gt Zymark Twister Plate Handler gt Twister The Zymark Twister Software launches Click Manual Control The software displays the Manual Control dialog box Click stack 4 Rotary Positioning Click 4 Rotary Adjustment 0 j i The plate handler arm moves over the input stack Click Find Plate If the adjustment was successful the plate handler arm will lower upon the plate until the plate detector switch engages confirming the presence of the plate If plate handler arm emits a grinding sound adjust the plate sensor switch as follows a From the Zymark Twister Software click Vertical Home to raise the plate handler arm b Turn the thumb screw in the Down direction 10 steps c Repeat step 6 until the plate handler arm successfully detects the plate Click Close Gripper then click Vertical Home If the adjustment was successful the plate ha
280. uttons and check boxes Working with windows Opening and closing resizing and repositioning scrolling understanding the active window Using the Microsoft Windows Finding files and creating folders hierarchical file system Getting Started 3 3 Conventions Used in This manual uses the following conventions to convey information This Manual Convention Definition Examples gt This symbol is used to convey a From the Start menu select command or directory path in Programs gt SDS 2 0 gt SDS 2 0 Men News epee yee Navigate to the Program Files gt Applied Biosystems gt SDS 2 0 gt Templates directory Bold text Bold text appearing with From the File menu select Save procenten cor E ands TONE The Detector Manager dialog box opens text as it appears on the screen How This Manualis_ This manual contains the following chapters and supporting appendices Organized 3 4 Getting Started Chapter Appendix Content 1 Safety Explains information on ABI PRISM 7900HT Sequence Detection System safety 2 Product Overview Describes the components of the ABI PRISM 7900HT Sequence Detection System and its software 3 Getting Started Introduces and explains how to use this manual and the SDS software 4 Run Setup and Explains how to create and run plate documents on the Basic Operation ABI PRISM 7900HT Sequence Detection
281. ystems C Program Files Applied Biosystems S Instrument Control S84N00LQ04 S84N00LR00 S84NO00LO 384N00L02L 384N00LR3A S84NO0LRA5 384NO00LRAD8 S84NO008HKE 384N0257M0 384N00LQGC SB4NO0LR J 384N00LQRD SB4N025075 S84N00LQN 384N016N9 Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Read Plate Queue must contain plate documents for all plates to be run Robot s Active Stacks IV Stacki 7 Stack2 7 Stack3 7 Stack 4 Open Close I Restack when finished Online Prepare and load the plates onto the plate handler as explained on page 4 36 4 34 Run Setup and Basic Operation Adding Plates to the To add plate documents to the plate queue Plate Queue Step Action 1 From the File menu of the Automation Controller Software select Add Plates The software displays the Open dialog box 2 From the Look in text field navigate to the directory containing the file or files of interest 3 While pressing and holding the Ctrl key click the plate document file s to add to the plate queue The software highlights selected files IMPORTANT A plate document must contain a bar code before it can be added to the plate queue See page 4 19 for more information on configuring a plate document with bar code information 4 Click Open The Automation Co
282. yzed each time they are opened The SDS software can print a report of the analyzed data containing individual or multiple elements of the plate document To print a report of the plate document data Step Action 1 From the File menu select Print Report 2 From the Print Report dialog box click the help button _ for instructions on setting up previewing and printing the report Exporting Plate Document Data as a Tab Delimited Text File The SDS software can export raw or analyzed data in tab delimited txt format for all or a select group of wells on a plate document The exported files are compatible with most spreadsheet applications and programs that can read tab delimited text To export run data as a tab delimited text file choose one of the following for further instructions See Exporting Plate Document Data on page B 9 Click the help button 7 within the table view Exporting Plots as Graphics The SDS software can export most panes and plots of the plate document as JPEG Joint Photographic Experts Group graphic files The JPEG file format is compatible with most word processing and spreadsheet applications and can be incorporated directly into HTML documents for viewing by most web browser software To export a plot as a graphic file see Exporting Graphics on page B 8 or click the help button within the plot of interest for further instructions Real Time An

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