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

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Designing TaqMan Assays C In This Appendix This appendix discusses the following topics: C 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 Development Program Identify Target <strong>Sequence</strong>(s) C-2 Designing TaqMan Assays To develop custom TaqMan 5´ nuclease assays: 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 A target template is a DNA, cDNA, RNA, or plasmid containing the nucleotide sequence of interest. For optimal results, the target template should meet the following requirements: ♦ 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.
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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
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Step 2 - Applying Detectors and Mar
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Creating an Allelic Discrimination
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Step 3 - Configuring the Plate Docu
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Step 4 - Programming the Plate Docu
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To create a method for the absolute
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Step 5 - Saving the Plate Document
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Step 7 - Applying Sample and Plate
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Section: Running an Individual Plat
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Preparing and Running a Single Plat
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Operating the 7900HT Instrument Usi
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After the Run Analyzing the Run Dat
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Section: Running Multiple Plates Us
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Adding a Plate Document to the Plat
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To create plate documents from a te
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Adding Plates to the Plate Queue Re
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Loading Plates To load plates onto
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End-Point Analysis 5 In This Chapte
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Section: Allelic Discrimination In
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Algorithmic Manipulation of Raw All
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Before You Begin Using SDS Online H
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Analyzing a Completed Allelic Discr
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Calling Allele Types To call allele
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Scrutinizing the Allele Calls To an
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After the Analysis Changing the Pla
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Real-Time Runs on the 7900HT Instru
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Overview About Absolute Quantificat
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Before You Begin Using SDS Online H
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Analyzing the Run Data Configuring
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Setting the Baseline and Threshold
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Eliminating Outliers For any PCR, e
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Viewing the Standard Curve 6-14 Rea
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6-16 Real-Time Analysis
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Overview About Dissociation Curve A
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Analysis Checklist WhereYouArein th
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Determining T m Values for the Anal
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After the Analysis Changing the Pla
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Recommended Maintenance Schedule Ma
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Replacing the Sample Block When to
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7-6 System Maintenance To remove th
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7-8 System Maintenance To replace t
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7-10 System Maintenance To replace
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Cleaning the Sample Block Wells 7-1
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Prepare a Background Plate Document
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Extracting the Background 7-16 Syst
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Materials Required The following ma
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Extracting Pure Dye Information fro
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Constructing a Custom Pure Dye Plat
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Verifying Instrument Performance Us
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Preparing and Running an RNase P Pl
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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
Page 171 and 172: Aligning the Fixed-Position Bar Cod
Page 173 and 174: 7-42 System Maintenance To position
Page 175 and 176: 7-44 System Maintenance
Page 177 and 178: General Computer Maintenance Mainte
Page 179 and 180: Maintaining the SDS software Admini
Page 181 and 182: Troubleshooting Table 8-2 Troublesh
Page 183 and 184: Low Precision or Irreproducibility
Page 185 and 186: Writing on the Reaction Plates Fluo
Page 187 and 188: Background Runs Background Troubles
Page 189 and 190: Pure Dye Runs Pure Dye Troubleshoot
Page 191 and 192: 8-12 Troubleshooting Troubleshootin
Page 193 and 194: Software and 7900HT Instrument Trou
Page 195 and 196: 8-16 Troubleshooting Troubleshootin
Page 198: User Bulletins 9 9 About This Chapt
Page 201 and 202: Fluorescent-Based Chemistries Funda
Page 203 and 204: Fluorescence Detection and Data Col
Page 205 and 206: Normalization of Reporter Signals A
Page 207 and 208: Determining Initial Template Concen
Page 209 and 210: Calculating Threshold Cycles A-10 T
Page 212 and 213: Importing and Exporting Plate Docum
Page 214 and 215: Configuring the Setup Table File wi
Page 216 and 217: About the Setup Table File Format S
Page 218 and 219: Setup Table Elements (continued) Nu
Page 220 and 221: Exporting Plate Document Data Expor
Page 224 and 225: Design Probes and Primers The follo
Page 226 and 227: Design Tips for Allelic Discriminat
Page 228 and 229: Kits, Reagents and Consumables D In
Page 230 and 231: Consumables and Disposables The ABI
Page 232: TaqMan Pre-Developed Assays and Rea
Page 236: Contacting Technical Support F Serv
Page 239 and 240: G-2 Limited Warranty Statement No a
Page 241 and 242: A(continued) Automation Controller
Page 243 and 244: I(continued) installing plate adapt
Page 245 and 246: Q quantifying probes and primers C-
Page 248 and 249: Headquarters 850 Lincoln Centre Dri
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