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

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Materials Required The following materials are required to perform the RNase P run: Preparing a RNase P Plate Document Material Part Number TaqMan RNase P Instrument Verification Plate Centrifuge, with plate adaptor See page D-4 To prepare a plate document for the RNase P plate: Step Action 1 Remove the TaqMan RNase P Instrument Verification Plate from the freezer and allow it to thaw to room temperature. 2 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 Template 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 a name 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 drop-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 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: where: 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. [ ( CopyUnk1) – 3( σCopyUnk1) ] > CopyUnk2 [ ( ) – 3( σCopyUnk2) ] CopyUnk a 1 = Average Copy Number of Unknown #1 (10,000 replicate population) σ a CopyUnk1 = Standard Deviation of Unknown #1 (10,000 replicate population) CopyUnk a 1 = Average Copy Number of Unknown #2 (5000 replicate population) σ a CopyUnk2 = Standard Deviation of Unknown #2 (5000 replicate population) a. These values can easily be obtained from the experimental report window. Note Up to 6 wells from each replicate group in a 96-well TaqMan RNase P Instrument Verification Plate can be ignored to meet specification. Note Up to 10 wells from each replicate group in a 384-well TaqMan RNase P Instrument Verification Plate can be ignored to meet specification.
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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
Page 106: After the Analysis Changing the Pla
Page 109 and 110: Real-Time Runs on the 7900HT Instru
Page 111 and 112: Overview About Absolute Quantificat
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: Verifying Instrument Performance Us
Page 159 and 160: Adjusting the Sensitivity of the Pl
Page 161 and 162: Testing the Adjustment 7-30 System
Page 163 and 164: Aligning the Plate Handler When to
Page 165 and 166: 7-34 System Maintenance To align th
Page 167 and 168: Re-checking the Input Stack 1 7-36
Page 169 and 170: 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
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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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