Mapping 250K/500K SNP assay

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70 GeneChip ® Mapping 500K Assay Manual LOAD PCR PLATES ONTO THERMAL CYCLERS PCR protocols for the MJ Tetrad PTC-225 and Applied Biosystems thermal cyclers are different. See Table 4.17 and Table 4.18 below. Location Main Lab Procedure To load the plates and run the 500K PCR program: 1. Transfer the reaction plates to the Main Lab. 2. Ensure that the thermal cycler lids are preheated. The block should be at room temperature. 3. Load each reaction plate onto a thermal cycler. 4. Run the 500K PCR program. The program varies depending upon the thermal cyclers you are using. See Table 4.17 for Applied Biosystems thermal cyclers and Table 4.18 for Bio-Rad thermal cyclers. If using GeneAmp® PCR System 9700 thermal cyclers, be sure the blocks are silver or gold-plated silver. Do NOT use thermal cyclers with aluminum blocks. It is not easy to visually distinguish between silver and aluminum blocks.
chapter 4 | 96-Well Plate Protocol 71 Table 4.17 500K PCR Thermal Cycler Program for the GeneAmp® PCR System 9700 (silver or gold-plated silver blocks) 500K PCR Program for GeneAmp ® PCR System 9700 Temperature Time Cycles 94ºC 3 minutes 1X 94ºC 60ºC 30 sec 45 sec } 30X 68ºC 15 sec 68ºC 7 minutes 1X 4ºC HOLD (Can be held overnight) Volume: 100 µL Specify Maximum mode. Table 4.18 500K PCR Thermal Cycler Program for the MJ Tetrad PTC-225 500K PCR Program for MJ Tetrad PTC-225 Temperature Time Cycles 94ºC 3 minutes 1X 94ºC 60ºC 68ºC 30 sec 30 sec 15 sec } 30X 68ºC 7 minutes 1X 4ºC HOLD (Can be held overnight) Volume: 100 µL Use Heated Lid and Calculated Temperature
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GeneChip ® Mapping 500K Assay Manu
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Contents CHAPTER 1 Overview 1 ABOUT
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contents v Equipment and Consumable
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contents vii Reagents Required 118
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contents ix CHAPTER 8 Troubleshooti
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contents xi STEP 4: PCR 257 Reagent
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Chapter 1 Overview
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About This Manual This manual is a
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chapter 1 | Overview 5 LD patterns
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Figure 1.1 GeneChip ® Mapping Assa
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chapter 1 | Overview 9 Gilman, B.,
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chapter 1 | Overview 11 23. Schaid,
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chapter 1 | Overview 13 35. Hu, N.,
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chapter 1 | Overview 15 50. Bignell
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chapter 1 | Overview 17 64. Gabriel
Page 31 and 32: Chapter 2 Laboratory Setup
Page 33 and 34: Introduction to Laboratory Setup Pl
Page 35 and 36: Main Lab Pre-PCR Clean Room chapter
Page 37 and 38: Chapter 3 Genomic DNA General Requi
Page 39 and 40: Introduction This chapter describes
Page 41 and 42: Sources of Human Genomic DNA chapte
Page 43 and 44: chapter 3 | Genomic DNA General Req
Page 45 and 46: Chapter 4 96-Well Plate Protocol
Page 47 and 48: ABOUT THIS PROTOCOL 96-Well Plate P
Page 49 and 50: chapter 4 | 96-Well Plate Protocol
Page 51 and 52: PREPARING THE WORK AREA FOR EACH ST
Page 53 and 54: PROGRAM YOUR THERMAL CYCLERS chapte
Page 55 and 56: EQUIPMENT AND CONSUMABLES REQUIRED
Page 57 and 58: ALIQUOTING PREPARED GENOMIC DNA To
Page 61 and 62: Negative Controls chapter 4 | 96-We
Page 63 and 64: Table 4.7 Nsp I Digestion Master Mi
Page 65 and 66: Stage 3: Ligation ABOUT THIS STAGE
Page 67 and 68: REAGENTS REQUIRED chapter 4 | 96-We
Page 71 and 72: DILUTE THE SAMPLES To dilute the sa
Page 73 and 74: Stage 4: PCR ABOUT THIS STAGE chapt
Page 77 and 78: About Controls chapter 4 | 96-Well
Page 79 and 80: B P1 P2 P3 Figure 4.2 Transferring
Page 81: Table 4.16 PCR Master Mix ADD PCR M
Page 85 and 86: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 91 and 92: POOL THE PCR PRODUCTS chapter 4 | 9
Page 101 and 102: Figure 4.5 Loading Optical Plates w
Page 103 and 104: Process Control Metrics Evaluate th
Page 105 and 106: Table 4.24 PROBLEM: Average Sample
Page 111 and 112: IMPORTANT INFORMATION ABOUT THIS ST
Page 113 and 114: Figure 4.6 Work Area Set Up for Sta
Page 119 and 120: Stage 8: Labeling ABOUT THIS STAGE
Page 127 and 128: LOCATION AND DURATION • Main Lab
Page 129 and 130: Hybridizing Samples Using Heat Bloc
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chapter 4 | 96-Well Plate Protocol
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Table 4.37 Hybridization Master Mix
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Load the Samples onto Arrays chapte
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Figure 4.8 Applying Tough-Spots ®
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Chapter 5 Washing, Staining, and Sc
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Introduction This chapter contains
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Reagent Preparation Wash A: Non-Str
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Experiment and Fluidics Station Set
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chapter 5 | Washing, Staining, and
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HANDLING THE GENECHIP ® PROBE ARRA
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Chapter 6 Fluidics Station Care and
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Introduction This chapter provides
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chapter 6 | Fluidics Station Care a
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White Clamp Figure 6.7 Releasing pe
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Figure 6.8 Troubleshooting decision
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PROBLEMS AND SOLUTIONS Table 6.3 Co
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Table 6.3 (Continued) Common error
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Table 6.4 (Continued) Common Error
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Table 6.5 (Continued) Other Problem
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Chapter 7 Analysis Workflow
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Introduction Software Requirements
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Figure 7.2 GTYPE main window with t
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Figure 7.3 Dynamic Model Mapping Al
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Table 7.1 Dynamic Model Mapping Alg
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FILE SETS chapter 7 | Analysis Work
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chapter 7 | Analysis Workflow 191 3
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NETAFFX SNP ANNOTATION chapter 7 |
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CALL RATE chapter 7 | Analysis Work
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chapter 7 | Analysis Workflow 197 G
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chapter 7 | Analysis Workflow 199 F
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chapter 7 | Analysis Workflow 201 S
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B2 OLIGO PERFORMANCE chapter 7 | An
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chapter 7 | Analysis Workflow 205 3
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chapter 7 | Analysis Workflow 207 W
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Chapter 8 Troubleshooting
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Assay Recommendations 211 Genotypin
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chapter 8 | Troubleshooting 213 10.
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Problem Likely Cause Solution Faint
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Problem Likely Cause Solution Posit
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Table 8.2 PROBLEM: Average Sample O
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When to Contact Technical Support c
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Appendix A Reagents, Equipment, and
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Reagents, Equipment, and Consumable
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appendix A | Reagents, Equipment, a
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Consumables Required GENECHIP ® AR
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Supplier Contact List Table A.10 Su
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Appendix B Thermal Cycler Programs
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ABOUT THIS APPENDIX 500K DIGEST 500
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500K FRAGMENT 500K LABEL 500K HYB a
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Appendix C Low Throughput Protocol
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Introduction The Affymetrix GeneChi
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BEFORE YOU BEGIN appendix C | Low T
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STEP 2: Restriction Enzyme Digestio
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DIGESTION PROCEDURE PRE-PCR CLEAN A
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STEP 3: Ligation REAGENTS AND EQUIP
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PCR STAGING AREA appendix C | Low T
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STEP 4: PCR REAGENTS AND EQUIPMENT
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PCR PROCEDURE PRE-PCR CLEAN ROOM ap
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MAIN LAB appendix C | Low Throughpu
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STEP 5: PCR Purification and Elutio
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appendix C | Low Throughput Protoco
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STEP 7: Fragmentation REAGENTS AND
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FRAGMENTATION PROCEDURE 1. Pre-heat
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LABELING PROCEDURE MAIN LAB appendi
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STEP 9: Target Hybridization REAGEN
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HYBRIDIZATION PROCEDURE appendix C
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Appendix D Reagents, Instruments, a
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Appendix D
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appendix D | Reagents, Instruments,
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* Adaptor, Nsp 5' ATTATGAGCACGACAGA
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Table D.4 Reagents Supplied by Affy
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Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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