ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

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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 quantification 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 T ) values for each sample (see page A-10). The software then computes a standard curve from the C T values of the diluted standards and extrapolates absolute quantities for the unknown samples based on their C T 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 transformations of sequence detection data. C T 28 26 24 22 Standard Curve Plot 20 1.0 E+2 1.0 E+3 1.0 E+4 1.0 E+5 Quantity (LogN initial concentration) Unknowns 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.
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ABI PRISM ® 7900HT Sequence Detect
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© Copyright 2001, Applied Biosyste
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iv Section:PlateDocumentSetup......
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vi A Theory of Operation Fluorescen
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Attention Words and Warning Labels
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About Waste Profiles A waste profil
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Safe Instrument Use Before Operatin
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Product Overview 2 In This Chapter
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Section: Getting to Know the Hardwa
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Interchangeable Thermal Cycler Bloc
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Computer Description The computer c
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Zymark Twister Microplate Handler D
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Instrument Connections Electrical C
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Section: Getting to Know the Softwa
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Managing Sequence Detection System
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Getting Started 3 In This Chapter T
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Using the SDS Software Online Help
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Turning on the ABI PRISM 7900HT Seq
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Using the SDS Software Workspace La
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Using the General Toolbar Using the
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Basic Software Skills Tutorial Abou
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Exercise 3: Opening a Plate Documen
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Lesson 2: Viewing and Resizing Pane
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Exercise 2: Selecting Multiple Well
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Lesson 4: Using the Hand-Held Bar C
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Using SDS Plate Documents Using Mul
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Run Setup and Basic Operation 4 In
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Setup Checklists Experiments/Runs P
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Section: Plate Document Setup In Th
Page 60 and 61: Step 2 - Applying Detectors and Mar
Page 62 and 63: Creating an Allelic Discrimination
Page 64 and 65: Step 3 - Configuring the Plate Docu
Page 66 and 67: Step 4 - Programming the Plate Docu
Page 68 and 69: To create a method for the absolute
Page 70 and 71: Step 5 - Saving the Plate Document
Page 72 and 73: Step 7 - Applying Sample and Plate
Page 74 and 75: Section: Running an Individual Plat
Page 76 and 77: Preparing and Running a Single Plat
Page 78 and 79: Operating the 7900HT Instrument Usi
Page 80 and 81: After the Run Analyzing the Run Dat
Page 82 and 83: Section: Running Multiple Plates Us
Page 84 and 85: Adding a Plate Document to the Plat
Page 86 and 87: To create plate documents from a te
Page 88 and 89: Adding Plates to the Plate Queue Re
Page 90 and 91: Loading Plates To load plates onto
Page 92 and 93: End-Point Analysis 5 In This Chapte
Page 94 and 95: Section: Allelic Discrimination In
Page 96 and 97: Algorithmic Manipulation of Raw All
Page 98 and 99: Before You Begin Using SDS Online H
Page 100 and 101: Analyzing a Completed Allelic Discr
Page 102 and 103: Calling Allele Types To call allele
Page 104 and 105: Scrutinizing the Allele Calls To an
Page 106: After the Analysis Changing the Pla
Page 109: Real-Time Runs on the 7900HT Instru
Page 113 and 114: Before You Begin Using SDS Online H
Page 115 and 116: Analyzing the Run Data Configuring
Page 117 and 118: Setting the Baseline and Threshold
Page 119 and 120: Eliminating Outliers For any PCR, e
Page 121 and 122: Viewing the Standard Curve 6-14 Rea
Page 123 and 124: 6-16 Real-Time Analysis
Page 125 and 126: Overview About Dissociation Curve A
Page 127 and 128: Analysis Checklist WhereYouArein th
Page 129 and 130: Determining T m Values for the Anal
Page 131 and 132: After the Analysis Changing the Pla
Page 133 and 134: Recommended Maintenance Schedule Ma
Page 135 and 136: Replacing the Sample Block When to
Page 137 and 138: 7-6 System Maintenance To remove th
Page 139 and 140: 7-8 System Maintenance To replace t
Page 141 and 142: 7-10 System Maintenance To replace
Page 143 and 144: Cleaning the Sample Block Wells 7-1
Page 145 and 146: Prepare a Background Plate Document
Page 147 and 148: Extracting the Background 7-16 Syst
Page 149 and 150: Materials Required The following ma
Page 151 and 152: Extracting Pure Dye Information fro
Page 153 and 154: Constructing a Custom Pure Dye Plat
Page 155 and 156: Verifying Instrument Performance Us
Page 157 and 158: Preparing and Running an RNase P Pl
Page 159 and 160: Adjusting the Sensitivity of the Pl
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Testing the Adjustment 7-30 System
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Aligning the Plate Handler When to
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7-34 System Maintenance To align th
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Re-checking the Input Stack 1 7-36
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Defining the Positions of the Remai
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Aligning the Fixed-Position Bar Cod
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7-42 System Maintenance To position
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7-44 System Maintenance
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General Computer Maintenance Mainte
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Maintaining the SDS software Admini
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Troubleshooting Table 8-2 Troublesh
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Low Precision or Irreproducibility
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Writing on the Reaction Plates Fluo
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Background Runs Background Troubles
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Pure Dye Runs Pure Dye Troubleshoot
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8-12 Troubleshooting Troubleshootin
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Software and 7900HT Instrument Trou
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8-16 Troubleshooting Troubleshootin
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User Bulletins 9 9 About This Chapt
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Fluorescent-Based Chemistries Funda
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Fluorescence Detection and Data Col
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Normalization of Reporter Signals A
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Determining Initial Template Concen
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Calculating Threshold Cycles A-10 T
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Importing and Exporting Plate Docum
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Configuring the Setup Table File wi
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About the Setup Table File Format S
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Setup Table Elements (continued) Nu
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Exporting Plate Document Data Expor
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Designing TaqMan Assays C In This A
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Design Probes and Primers The follo
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Design Tips for Allelic Discriminat
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Kits, Reagents and Consumables D In
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Consumables and Disposables The ABI
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TaqMan Pre-Developed Assays and Rea
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Contacting Technical Support F Serv
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G-2 Limited Warranty Statement No a
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A(continued) Automation Controller
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I(continued) installing plate adapt
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Q quantifying probes and primers C-
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Headquarters 850 Lincoln Centre Dri
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