Mapping 250K/500K SNP assay

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40 GeneChip ® Mapping 500K Assay Manual THERMAL CYCLERS, PLATES AND PLATE SEALS The Mapping 500K 96-well plate protocol has been optimized using the following thermal cyclers, reaction plates and adhesive film. Use only the PCR plate, adhesive film and thermal cyclers listed in Table 4.1. Using other PCR plates and film that are incompatible with these thermal cyclers can result in crushed tubes, loss of sample, or poor results. Table 4.1 Thermal Cyclers, PCR Plate and Adhesive Film Optimized for Use With the 500K 96-well Plate Protocol Area Thermal Cyclers Validated for Use Pre-PCR PCR and Post-PCR 2720 Thermal Cycler or GeneAmp ® PCR System 9700 by Applied Biosystems MJ Tetrad PTC-225 by Bio-Rad GeneAmp ® PCR System 9700 by Applied Biosystems (silver block or goldplated silver block) MJ Tetrad PTC-225 by Bio-Rad Plate Film Multiplate 96-Well Unskirted PCR Plates Bio-Rad, P/N MLP-9601 Multiplate 96-Well Unskirted PCR Plates Bio-Rad, P/N MLP-9601 MicroAmp ® Clear Adhesive Films Applied Biosystems, P/N 4306311 MicroAmp ® Clear Adhesive Films Applied Biosystems, P/N 4306311
PROGRAM YOUR THERMAL CYCLERS chapter 4 | 96-Well Plate Protocol 41 The Mapping 500K 96-well plate protocol includes the thermal cycler programs listed below. Before you begin processing samples, enter and store these programs on the thermal cyclers that will be used for this protocol. Thermal cycler program details are listed in Appendix B, Thermal Cycler Programs. Table 4.2 Thermal Cycler Programs Required for the 96-well Plate Protocol Program Name # of Thermal Cyclers Required (based on 96 sample throughput) Laboratory 500K Digest 1 Pre-PCR Clean Area 500K Ligate 1 Pre-PCR Clean Area 500K PCR 3 Main Lab 500K Fragment 1 Main Lab 500K Label 1 Main Lab 500K Hyb 1 Main Lab CUTTING STRIP TUBES FROM PLATES Strip tubes are required for many of the steps in the 96-well plate protocol. The Mapping 500K 96-well plate protocol has been optimized using strips of tubes that have been cut from the Bio-Rad plates recommended in Table 4.1 on page 40 and in Appendix A, Reagents, Equipment, and Consumables Required for 96-Well Plate Protocol.
Page 1 and 2: GeneChip ® Mapping 500K Assay Manu
Page 3 and 4: Contents CHAPTER 1 Overview 1 ABOUT
Page 5 and 6: contents v Equipment and Consumable
Page 7 and 8: contents vii Reagents Required 118
Page 9 and 10: contents ix CHAPTER 8 Troubleshooti
Page 11 and 12: contents xi STEP 4: PCR 257 Reagent
Page 13 and 14: Chapter 1 Overview
Page 15 and 16: About This Manual This manual is a
Page 17 and 18: chapter 1 | Overview 5 LD patterns
Page 19 and 20: Figure 1.1 GeneChip ® Mapping Assa
Page 21 and 22: chapter 1 | Overview 9 Gilman, B.,
Page 23 and 24: chapter 1 | Overview 11 23. Schaid,
Page 25 and 26: chapter 1 | Overview 13 35. Hu, N.,
Page 27 and 28: chapter 1 | Overview 15 50. Bignell
Page 29 and 30: 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: PREPARING THE WORK AREA FOR EACH ST
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 and 82: Table 4.16 PCR Master Mix ADD PCR M
Page 85 and 86: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 87 and 88: EQUIPMENT AND CONSUMABLES REQUIRED
Page 91 and 92: POOL THE PCR PRODUCTS chapter 4 | 9
Page 95 and 96: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 101 and 102: Figure 4.5 Loading Optical Plates w
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Process Control Metrics Evaluate th
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Table 4.24 PROBLEM: Average Sample
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WHAT YOU CAN DO NEXT chapter 4 | 96
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EQUIPMENT AND CONSUMABLES REQUIRED
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IMPORTANT INFORMATION ABOUT THIS ST
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Figure 4.6 Work Area Set Up for Sta
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chapter 4 | 96-Well Plate Protocol
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Stage 8: Labeling ABOUT THIS STAGE
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REAGENTS REQUIRED chapter 4 | 96-We
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LOCATION AND DURATION • Main Lab
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Hybridizing Samples Using Heat Bloc
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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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