Mapping 250K/500K SNP assay

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Introduction 286 This section describes the reagents, supplies and equipment required to implement the GeneChip® Mapping 500K Low Throughput Protocol. The first section describes the Affymetrix GeneChip® Mapping 250K Assay Kits for Nsp and Sty (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), which contains critical reagents and reagent components specifically developed and/or optimized for the GeneChip Mapping 500K Assay, and controls that are integrated for use with the GeneChip® Mapping 500K Array Set. This section also indicates the Affymetrix equipment – scanner, fluidics station, hybridization oven - and GeneChip Mapping 250K arrays required for this assay. The second section describes the reagents, supplies and instruments that are required, but not supplied directly by Affymetrix. The products and vendors listed in this section have been tested at Affymetrix during the development of the product – other products and vendors have not been verified at Affymetrix. In some cases, we have seen lower performance when using non-recommended vendors.
appendix D | Reagents, Instruments, and Supplies Required for Low Throughput Protocol 287 Reagents and Instruments Required Table D.1 Reagents Supplied by Affymetrix - 30 Reaction Kit The following reagents and equipment are required to process the GeneChip Mapping 500K Array Set. Affymetrix GeneChip ® Mapping 250K Nsp Assay Kit P/N 900766 This kit includes sufficient reagent for 30 arrays. Component Volume Concentration Description Box # Adaptor, Nsp I 25 µL 50 µM Two annealed oligonucleotides, specific for ligation to Nsp restriction site. PCR Primer, 002 450 µL 100 µM PCR primer, to amplify ligated genomic DNA Reference Genomic DNA, 103 GeneChip ® Fragmentation Reagent 30 µL 50 ng/µL Human genomic DNA (single source) 2 25 µL See label on tube DNase I enzyme, formulated to fragment purified PCR amplicons 10X Fragmentation Buffer 250 µL 10X Buffer for fragmentation reaction 3 GeneChip ® DNA Labeling Reagent (30 mM) Terminal Deoxynucleotidyl Transferase 5X Terminal Deoxynucleotidyl Transferase Buffer Oligo Control Reagent, 0100 (OCR, 0100) 60 µL 30 mM Biotin-labeled reagent for end-labeling fragmented PCR amplicons 105 µL 30 U/µL Enzyme used to end-label fragmented PCR amplicons with the GeneChip ® DNA Labeling Reagent (30 mM) 420 µL 5X Buffer for labeling reactio 3 60 µL See protocol Chapter 4 (high throughput) or Appendix C (low throughput) Mixture of five biotin-labeled oligonucleotides, which hybridize to control regions (gridding controls and array controls) on the GeneChip ® Mapping 250K Arrays. 1 1 3 3 3 3
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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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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 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 301 and 302: * Adaptor, Nsp 5' ATTATGAGCACGACAGA
Page 303 and 304: Table D.4 Reagents Supplied by Affy
Page 305 and 306: appendix D | Reagents, Instruments,
Page 309 and 310: Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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