Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

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Chapter 4Operating the InstrumentPreventing ContaminationContaminationand the5´ NucleaseAssayGeneral PCRPracticesPCR using the 5´ nuclease assay requires special laboratory practices to avoid falsepositive amplifications (Kwok and Higuchi, 1989). The assay’s logarithmicamplifications can potentially amplify a single DNA molecule (Saiki et al., 1985;Mullis and Faloona, 1987).Observe the following guidelines when assembling optical plates or loadingTaqMan ® Low Density Arrays for use on the 7900HT instrument:• Wear a clean lab coat (not previously worn while handling amplified PCR productsor used during sample preparation) and clean gloves when preparing samples forPCR amplification.• Change gloves whenever you suspect they are contaminated.• Maintain separate areas, dedicated equipment, and supplies for:– Sample preparation– PCR setup– PCR amplification– Analysis of PCR products• Never bring amplified PCR products into the PCR setup area.• Open and close all sample tubes carefully. Try not to splash or spray PCR samples.• Keep reactions and components capped as much as possible.• Use positive-displacement pipets or aerosol-resistant pipet tips.• Clean lab benches and equipment with 10% bleach solution.Compatible ConsumablesConsumables forUse withthe 7900HTInstrumentTable 4-1 Compatible ConsumablesConsumable TypeUse these…Compatible SealsNot these…ABI PRISM 384-Well OpticalReaction Plates• ABI PRISM Optical Adhesive Covers(quantity 100)• Optical Adhesive Covers (quantity 25)384-Well Septa4-6 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm
Compatible ConsumablesTable 4-1Compatible ConsumablesConsumable TypeUse these…Compatible SealsNot these…ABI PRISM 96-WellOptical ReactionPlatesOptical 96-WellFast ThermalCycling PlatesTaqMan ® LowDensity Arrays• Compression Pads:– Standard pad for manual operation– ABI PRISM Snap-On CompressionPad (reusable)• 96-Well Plate Seals:– ABI PRISM Optical Adhesive Covers(quantity 100)– Optical Adhesive Covers(quantity 25)– ABI PRISM Optical Caps(flat caps only)• ABI PRISM Optical Adhesive Covers(quantity 100)• Optical Adhesive Covers (quantity 25)None (self sealing)• ABI PRISM Optical Caps(round caps)• 96-Well SeptaNone (self sealing)Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide 4-7DRAFTSeptember 1, 2004 11:39 am, CH_Basic.fm
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Applied Biosystems7900HT Fast Real-
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ContentsPrefaceHow to Use This Guid
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Chapter 4Operating the InstrumentNo
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Chapter 7Maintaining the Instrument
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Appendix D Theory of OperationFluor
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PrefaceHow to Use This GuidePurpose
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Safety and EMC Compliance Informati
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Symbols on InstrumentsSymbols on In
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General Instrument SafetyGeneral In
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Chemical Waste SafetyChemical Safet
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Physical Hazard SafetyOvervoltageRa
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Workstation SafetyWorkstation Safet
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Product Overview 11In This Chapter
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7900HT FAST Real-Time PCR SystemSec
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7900HT InstrumentInterchangeableThe
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7900HT FAST Real-Time PCR SystemBar
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Compatible ConsumablesCompatible Co
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Instrument ConnectionsFixed-Positio
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Section 1.2 Getting to Know the Sof
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SDS Software Related Files and Form
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7900HT FAST Real-Time PCR SystemMan
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Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
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Section 1.3 SDS Enterprise Database
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* + Enter* +Enter3Down UpNumLockSys
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About the SDS Enterprise Database S
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Esc F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
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Getting Started 22In This Chapter G
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Getting StartedUsing the SDSSoftwar
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GR23967900HTPowering On the 7900HT
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Using the SDS SoftwareUsing the SDS
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Using the SDS SoftwareAbout theMess
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Basic Software Skills TutorialNotes
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Basic Software Skills TutorialExerc
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Basic Software Skills TutorialExpor
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Page 73 and 74: Basic Software Skills TutorialLesso
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Page 81 and 82: Using SDS Plate DocumentsUsing SDS
Page 83 and 84: Using SDS Plate DocumentsWell Inspe
Page 85 and 86: Preparing a Run 33In This Chapter N
Page 87 and 88: Notes for Database UsersDescription
Page 89 and 90: Workflow OverviewWorkflow OverviewE
Page 91 and 92: Workflow OverviewAllelicDiscriminat
Page 93 and 94: Step 1 - Creating a Plate DocumentS
Page 95 and 96: Step 2 - Applying Detectors and Mar
Page 101 and 102: Step 3 - Configuring the Plate Docu
Page 103 and 104: Step 5 - Programming the Plate Docu
Page 109 and 110: Step 6 - Saving the Plate Document
Page 111 and 112: Step 7 - Creating a Plate Document
Page 113 and 114: Step 9 - Running the Plate on the 7
Page 115 and 116: Operating the Instrument 44In This
Page 117 and 118: Notes for Database UsersDescription
Page 119: Section 4.1 Consumable PreparationS
Page 123 and 124: Preparing Optical Plates for Usea.
Page 125 and 126: Preparing TaqMan Low Density Arrays
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Page 139 and 140: Running a Single Plate (Using the S
Page 141 and 142: Running a Single Plate (Using the S
Page 143 and 144: After the RunAfter the RunUser Acce
Page 145 and 146: Section 4.3 Automated OperationSect
Page 147 and 148: Operating the Software with an SDS
Page 149 and 150: Operating the Software without an S
Page 155 and 156: Running Plates Using the Automation
Page 157 and 158: Running Plates Using the Automation
Page 159 and 160: Analyzing End-Point Data 55In This
Page 161 and 162: Notes for Database UsersNotes for D
Page 163 and 164: Section 5.1 Allelic DiscriminationS
Page 165 and 166: OverviewTable 5-1Signal and Sequenc
Page 167 and 168: Before You BeginBefore You BeginUsi
Page 169 and 170: Opening the Run DataOpening the Run
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Calling and Scrutinizing Allelic Di
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After the AnalysisSaving theResults
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Analyzing Real-Time Data 66In This
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Notes for Database UsersDescription
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Section 6.1 Absolute Quantification
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OverviewAlgorithmicManipulation ofR
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Analysis ChecklistAnalysis Checklis
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Analyzing the DataAnalyzing the Dat
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Viewing ResultsViewing ResultsViewi
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Section 6.2 Relative Quantification
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OverviewAlgorithmic Manipulation of
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OverviewAutomatic Outlier RemovalAb
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Getting StartedGetting StartedUsing
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Options for Analyzing Relative Quan
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Creating the Studyd. In the Plate Q
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Analyzing the Study• Manual Ct -
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Analyzing the StudySpecifying the A
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Viewing ResultsViewing ResultsDispl
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Viewing ResultsAbout DownArrowsDown
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After the AnalysisAfter the Analysi
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Section 6.3 Dissociation Curve Anal
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Before You BeginExample ResultsFigu
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Opening the Run DataOpening the Run
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Determining T m Values for the Anal
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Section 6.4 Procedure ReferenceSect
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Setting the Baseline and Threshold
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Eliminating Outlying AmplificationE
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Eliminating Outlying Amplification6
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After the AnalysisSaving theResults
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Maintaining the Instrument 77In Thi
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Notes for Database UsersDescription
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Section 7.1 Maintaining the 7900HT
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Replacing the Sample BlockMaterials
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Replacing the Sample Block9. Loosen
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7900HT FAST Real-Time PCR SystemRep
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Changing the Plate Adapter4. Place
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Decontaminating the Sample BlockCle
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Performing a Background RunPreparin
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Performing a Background RunAnalyzin
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Performing a Pure Dye RunAfter a ru
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Performing a Pure Dye Run• Fast 9
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Performing a Pure Dye RunAnalyzing
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Adding Custom Dyes to the Pure Dye
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Adding Custom Dyes to the Pure Dye
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Verifying Instrument PerformanceTab
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Verifying Instrument PerformancePre
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Section 7.2 Maintaining the Plate H
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Adjusting the Sensitivity of the Pl
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Adjusting the Sensitivity of the Pl
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7900HT FAST Real-Time PCR SystemAli
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Aligning the Plate Handler5. Using
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Aligning the Plate Handler7. Using
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Aligning the Plate Handler13. Click
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7900HT FAST Real-Time PCR SystemAli
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Aligning the Fixed-Position Bar Cod
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Section 7.3 Maintaining the Compute
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Maintaining the SDS softwareSeveral
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Troubleshooting 88In This Chapter T
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Troubleshooting TableTable 8-1Troub
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Low Precision or IrreproducibilityL
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Low Precision or IrreproducibilityD
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Background RunsBackground RunsBackg
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Pure Dye Runs6. Repeat step 4 until
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End-Point Runs (Allelic Discriminat
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Software and 7900HT InstrumentTable
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Zymark Twister Microplate Handler a
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TaqMan Low Density ArrayTable 8-6Tr
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Software ReferenceAAIn This Appendi
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Importing Plate Document Setup Tabl
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Setup Table Files1. File VersionDes
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Setup Table Files6. Detectors ListD
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Setup Table Files9. Markers List (A
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Setup Table FilesFormat(for a singl
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Setup Table FilesFormat(for a singl
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Setup Table FilesExample Code A-16A
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Exporting Plate Document Data4. In
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Operating the SDS Software from a C
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Using the Search ToolLogical Operat
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Connecting SDS Software to the Data
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Designing TaqMan Reagent-BasedAssay
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Assay Development Guidelines• For
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Design Tips for Allelic Discriminat
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Kits, Reagents, and ConsumablesCCIn
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Consumables and DisposablesConsumab
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Instrument Maintenance and Verifica
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Custom Oligonucleotide SynthesisTab
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Theory of OperationDDIn This Append
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Fluorescent-Based ChemistriesBasics
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Real-Time Data AnalysisPhase 2: Lin
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Real-Time Data AnalysisSignificance
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A similar equation for the endogeno
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Limited Warranty StatementEEWarrant
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BibliographyGeneral Paperson TaqMan
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Klein, D., Janda, P., Steinborn, R.
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Dolken, L., F. , Schuler, and G. ,
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AllelicDiscriminationVirtanen M., H
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Glossary.sdd.sdm.sds.sdt∆C T∆
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ackgroundbaselinecalibratorcDNA(com
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minor groovebinder (MGB)multicompon
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R nrunsessionsingle nucleotidepolym
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IndexSymbols- 6-31↑ 6-33Numerics7
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fluorescent, common sources 8-7isol
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adjusting step parameters 3-21confi
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sample block locking bar 7-8sample
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Worldwide Sales and SupportApplied
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