Mapping 250K/500K SNP assay

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* Adaptor, Sty 5' ATTATGAGCACGACAGACGCCTGATCT 3' 292 GeneChip ® Mapping 500K Assay Manual 3' AATACTCGTGCTGTCTGCGGACTAGAGWWCp 5' PCR Primer, 002 5' ATTATGAGCACGACAGACGCCTGATCT 3' Table D.5 Equipment and Software Supplied by Affymetrix Item Part Number GeneChip ® Fluidics Station 450* 00-0079 GeneChip ® Hybridization Oven 640* 800139 GeneChip ® Scanner 3000 7G* 00-0205 Affymetrix GeneChip ® Operating Software version 1.4* 690031 Affymetrix GeneChip ® Genotyping Analysis Software 4.0* 690051 GeneChip ® Human Mapping 250K Nsp Array* 520330 GeneChip ® Human Mapping 250K Sty Array* 520331 * Denotes critical reagents, equipment or supplies. Formulations or vendors not listed here have not been tested and verified at Affymetrix. In some cases, lower performance has been demonstrated by reagents from non-qualified vendors.
appendix D | Reagents, Instruments, and Supplies Required for Low Throughput Protocol 293 Table D.6 Reagents, Equipment and Supplies Not Supplied by Affymetrix Assay Step Reagents Equipment Genomic DNA Preparation Restriction Enzyme Digestion Reduced EDTA TE Buffer (10 mM Tris HCL, 0.1 mM EDTA, pH 8.0): TEKnova, P/N T0223 Reference Genomic DNA, 103 (50 ng/µL): Affymetrix * , P/N 900421, available in Box 2 of the 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 250 ng Genomic DNA 50 ng/µL working stock/array Sty I (10,000 U/mL): New England Biolabs (NEB), P/N R0500S (if running Sty Array) containing: NE Buffer 3: New England Biolabs (NEB), (P/N B7003S to order separately) BSA (Bovine Serum Albumin): New England Biolabs (NEB), (P/N B9001S to order separately) Nsp I (10,000 U/mL): New England Biolabs (NEB), P/N R0602L (if running Nsp Array) containing: NE Buffer 2: New England Biolabs (NEB), (P/N B7002S to order separately) H 2O (Molecular Biology Grade Water): BioWhittaker Molecular Applications/Cambrex, P/N 51200 96-well plate: Bio-Rad, P/N MLP-9601; or Applied Biosystems, P/N 403083 96-well Plate Clear Adhesive Films: Applied Biosystems, P/N 4306311 8-Tube Strips, thin-wall (0.2 mL): Bio-Rad, P/N TBS-0201; Strip of 8 caps: Bio-Rad, P/N TCS-0801 Thermal Cycler
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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
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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
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
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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 and 288: LABELING PROCEDURE MAIN LAB appendi
Page 289 and 290: STEP 9: Target Hybridization REAGEN
Page 291 and 292: HYBRIDIZATION PROCEDURE appendix C
Page 295 and 296: Appendix D Reagents, Instruments, a
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Page 309 and 310: Fragmentation and Labeling appendix
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