Mapping 250K/500K SNP assay

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136 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual Reagents and Materials Required The following reagents and materials are recommendations and have been tested and evaluated by Affymetrix scientists. Information and part numbers listed are based on U.S. catalog information. • Water, Molecular Biology Grade: BioWhittaker Molecular Applications/Cambrex, P/N 51200 • Distilled water: Invitrogen Life Technologies, P/N 15230147 • 20X SSPE (3 M NaCl, 0.2 M NaH 2PO 4, 0.02 M EDTA): BioWhittaker Molecular Applications/Cambrex, P/N 51214 • Anti-streptavidin antibody (goat), biotinylated: Vector Laboratories, P/N BA-0500; reconstitute according to product instructions • R-Phycoerythrin Streptavidin: Molecular Probes, P/N S-866 • 10% Surfact-Amps ® 20 (Tween-20): Pierce Chemical, P/N 28320 • Bleach (5.25% Sodium Hypochlorite): VWR Scientific, P/N 21899-504 (or equivalent) • Denhardt’s Solution, 50X concentrate: Sigma, P/N D2532 • MES hydrate: Sigma-Aldrich, P/N M5287 • MES Sodium Salt: Sigma-Aldrich, P/N M5057 • 5 M NaCl, RNase-free, DNase-free: Ambion, P/N 9760G Miscellaneous Supplies • Sterile, RNase-free, microcentrifuge vials, 1.5 mL: USA Scientific, P/N 1415-2600 (or equivalent) • Micropipettors, (P-2, P-20, P-200, P-1000): Rainin Pipetman ® (or equivalent) • Sterile-barrier pipette tips and non-barrier pipette tips •Tygon ® Tubing, 0.04” inner diameter: Cole-Parmer, P/N H-06418-04 • Tough-Spots ® , Label Dots: USA Scientific, P/N 9185-0000
Reagent Preparation Wash A: Non-Stringent Wash Buffer (6X SSPE, 0.01% Tween 20) For 1000 mL: 300 mL of 20X SSPE 1.0 mL of 10% Tween-20 699 mL of water Filter through a 0.2 µm filter. Store at room temperature. Wash B: Stringent Wash Buffer (0.6X SSPE, 0.01% Tween 20) For 1000 mL: 30 mL of 20X SSPE 1.0 mL of 10% Tween-20 969 mL of water Filter through a 0.2 µm filter. Store at room temperature. The pH should be 8. chapter 5 | Washing, Staining, and Scanning Arrays 137 Prepare Wash B in smaller quantities to avoid long term storage. The container must be sealed tightly to avoid changes in salt concentration due to evaporation. 0.5 mg/mL Anti-Streptavidin Antibody Resuspend 0.5 mg in 1 mL of water. Store at 4°C.
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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
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Chapter 2 Laboratory Setup
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Introduction to Laboratory Setup Pl
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Main Lab Pre-PCR Clean Room chapter
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Chapter 3 Genomic DNA General Requi
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Introduction This chapter describes
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Sources of Human Genomic DNA chapte
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chapter 3 | Genomic DNA General Req
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Chapter 4 96-Well Plate Protocol
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ABOUT THIS PROTOCOL 96-Well Plate P
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chapter 4 | 96-Well Plate Protocol
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PREPARING THE WORK AREA FOR EACH ST
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PROGRAM YOUR THERMAL CYCLERS chapte
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EQUIPMENT AND CONSUMABLES REQUIRED
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ALIQUOTING PREPARED GENOMIC DNA To
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Negative Controls chapter 4 | 96-We
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Table 4.7 Nsp I Digestion Master Mi
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Stage 3: Ligation ABOUT THIS STAGE
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REAGENTS REQUIRED chapter 4 | 96-We
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DILUTE THE SAMPLES To dilute the sa
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Stage 4: PCR ABOUT THIS STAGE chapt
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REAGENTS REQUIRED chapter 4 | 96-We
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About Controls chapter 4 | 96-Well
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B P1 P2 P3 Figure 4.2 Transferring
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Table 4.16 PCR Master Mix ADD PCR M
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WHAT YOU CAN DO NEXT chapter 4 | 96
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EQUIPMENT AND CONSUMABLES REQUIRED
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POOL THE PCR PRODUCTS chapter 4 | 9
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WHAT YOU CAN DO NEXT chapter 4 | 96
Page 97 and 98: REAGENTS REQUIRED chapter 4 | 96-We
Page 99 and 100: chapter 4 | 96-Well Plate Protocol
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 107 and 108: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 109 and 110: EQUIPMENT AND CONSUMABLES REQUIRED
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 121 and 122: REAGENTS REQUIRED chapter 4 | 96-We
Page 127 and 128: LOCATION AND DURATION • Main Lab
Page 129 and 130: Hybridizing Samples Using Heat Bloc
Page 135 and 136: Table 4.37 Hybridization Master Mix
Page 137 and 138: Load the Samples onto Arrays chapte
Page 139 and 140: Figure 4.8 Applying Tough-Spots ®
Page 145 and 146: Chapter 5 Washing, Staining, and Sc
Page 147: Introduction This chapter contains
Page 151 and 152: Experiment and Fluidics Station Set
Page 153 and 154: chapter 5 | Washing, Staining, and
Page 161 and 162: HANDLING THE GENECHIP ® PROBE ARRA
Page 165 and 166: Chapter 6 Fluidics Station Care and
Page 167 and 168: Introduction This chapter provides
Page 169 and 170: chapter 6 | Fluidics Station Care a
Page 179 and 180: White Clamp Figure 6.7 Releasing pe
Page 181 and 182: Figure 6.8 Troubleshooting decision
Page 183 and 184: PROBLEMS AND SOLUTIONS Table 6.3 Co
Page 185 and 186: Table 6.3 (Continued) Common error
Page 187 and 188: Table 6.4 (Continued) Common Error
Page 189 and 190: Table 6.5 (Continued) Other Problem
Page 191 and 192: Chapter 7 Analysis Workflow
Page 193 and 194: Introduction Software Requirements
Page 195 and 196: Figure 7.2 GTYPE main window with t
Page 197 and 198: 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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