Mapping 250K/500K SNP assay

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276 GeneChip ® Mapping 500K Assay Manual To expedite the aliquoting, the Labeling Master Mix can be first divided into 8 or 12 microtube strips and then dispensed into the wells of the plate with an 8- or 12-channel pipette. Pipet up and down several times to mix. Be sure to change tips between samples. 3. Seal the plate tightly with a plate cover. 4. Vortex the plate at medium speed for 2 seconds, and spin the plate at 2,000 rpm for 1 minute. 5. Run the 500K Label program: 500K Label Program Temperature Time 37ºC 4 hours 95ºC 15 minutes 4ºC Hold Make sure the plates/reaction tubes are securely sealed prior to running this program in order to minimize solution loss due to evaporation at denaturation step (95°C). 6. Spin the plate at 2,000 rpm for 1 minute after the labeling reaction. Samples can be stored at –20°C if not proceeding to next step.
STEP 9: Target Hybridization REAGENTS appendix C | Low Throughput Protocol 277 • 5 M TMACL (Tetramethyl Ammonium Chloride): Sigma, P/N T3411 • 10% Tween-20: Pierce, P/N 28320 (Surfact-Amps ® ); diluted to 3% in molecular biology grade water • MES hydrate SigmaUltra: Sigma, P/N M5287 • MES Sodium Salt: Sigma, P/N M5057 • DMSO: Sigma, P/N D5879 • EDTA: Ambion, P/N 9260G • Denhardt’s Solution: Sigma, P/N D2532 • HSDNA (Herring Sperm DNA): Promega, P/N D1815 • Human Cot-1 DNA ® : Invitrogen, P/N 15279-011 • Oligo Control Reagent, 0100 (OCR, 0100): Affymetrix, P/N 900541 for 30 Rxns and 900701 for 100 Rxns. Available in the Nsp and Sty GeneChip ® Mapping 250K Assay Kits: - Nsp 30 Rxn Kit P/N 900766 - Nsp 100 Rxn Kit P/N 900753 - Sty 30 Rxn Kit P/N 900765 - Sty 100 Rxn Kit P/N 900754 We have noticed increased Mapping 500K performance variability in the GeneChip ® Hybridization Oven 640 models (P/N 800138 or 800189) manufactured prior to 2001. We have not seen this increased variability in later Hybridization Oven 640 models with a serial number of 11215 or higher. Please check the serial number of your hybridization oven(s). If you have one of the older models with a number of 11214 or lower, please contact Affymetrix for an upgrade.
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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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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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POOL THE PCR PRODUCTS chapter 4 | 9
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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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IMPORTANT INFORMATION ABOUT THIS ST
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Figure 4.6 Work Area Set Up for Sta
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Stage 8: Labeling ABOUT THIS STAGE
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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
Page 237 and 238: Reagents, Equipment, and Consumable
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Page 245 and 246: Consumables Required GENECHIP ® AR
Page 247 and 248: Supplier Contact List Table A.10 Su
Page 249 and 250: Appendix B Thermal Cycler Programs
Page 251 and 252: ABOUT THIS APPENDIX 500K DIGEST 500
Page 253 and 254: 500K FRAGMENT 500K LABEL 500K HYB a
Page 255 and 256: Appendix C Low Throughput Protocol
Page 257 and 258: Introduction The Affymetrix GeneChi
Page 259 and 260: BEFORE YOU BEGIN appendix C | Low T
Page 261 and 262: STEP 2: Restriction Enzyme Digestio
Page 263 and 264: DIGESTION PROCEDURE PRE-PCR CLEAN A
Page 265 and 266: STEP 3: Ligation REAGENTS AND EQUIP
Page 267 and 268: PCR STAGING AREA appendix C | Low T
Page 269 and 270: STEP 4: PCR REAGENTS AND EQUIPMENT
Page 271 and 272: PCR PROCEDURE PRE-PCR CLEAN ROOM ap
Page 273 and 274: MAIN LAB appendix C | Low Throughpu
Page 275 and 276: STEP 5: PCR Purification and Elutio
Page 277 and 278: appendix C | Low Throughput Protoco
Page 279 and 280: STEP 7: Fragmentation REAGENTS AND
Page 281 and 282: FRAGMENTATION PROCEDURE 1. Pre-heat
Page 287: LABELING PROCEDURE MAIN LAB appendi
Page 291 and 292: HYBRIDIZATION PROCEDURE appendix C
Page 295 and 296: Appendix D Reagents, Instruments, a
Page 297 and 298: Appendix D
Page 299 and 300: appendix D | Reagents, Instruments,
Page 301 and 302: * Adaptor, Nsp 5' ATTATGAGCACGACAGA
Page 303 and 304: Table D.4 Reagents Supplied by Affy
Page 309 and 310: Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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