Mapping 250K/500K SNP assay

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248 GeneChip ® Mapping 500K Assay Manual Genomic DNA Preparation PREPARATION OF GENOMIC DNA REAGENTS The concentration of the genomic DNA should be determined and the working stocks diluted to 50 ng/µL using reduced EDTA TE buffer (0.1 mM EDTA, 10 mM Tris HCL, pH 8.0). Diluted genomic DNA can be aliquoted at this point. For high throughput assays (2 or more 96-well plates), make multiple replicates of each plate. An elevated EDTA level is not recommended as it may interfere with subsequent reactions. • Reduced EDTA TE Buffer (10 mM Tris HCL, 0.1 mM EDTA, pH 8.0):TEKnova, P/N T0223 STEP 1: Reagent Preparation and Storage The reagents necessary for the restriction digestion, ligation and PCR steps should be stored in the pre-PCR clean room (or area for the DNA template and free of PCR products) to minimize cross contamination between samples. To avoid re-entering the pre-PCR clean room after entering either the PCR-Staging Room or the Main Lab, Affymetrix recommends aliquoting each of the reagents in the pre-PCR clean room before starting the rest of the experiment.
STEP 2: Restriction Enzyme Digestion REAGENTS AND EQUIPMENT appendix C | Low Throughput Protocol 249 • 250 ng Genomic DNA per array working stock: 50 ng/µL • Sty I (10,000 U/mL): New England Biolabs (NEB), P/N R0500S containing: - NE Buffer 3: New England Biolab (NEB), (P/N B7003S to order separately) - BSA (Bovine Serum Albumin): New England Biolab (NEB), (P/N B9001S to order separately) • Nsp I (10,000 U/mL): New England Biolab (NEB), P/N R0602L containing: - NE Buffer 2: New England Biolab (NEB), (P/N B7002S to order separately) - BSA (Bovine Serum Albumin): New England Biolab (NEB), (P/N B9001S to order separately) •H2O (Molecular Biology Grade Water): BioWhittaker Molecular Applications/Cambrex, P/N 51200 • 96-well plate: Bio-Rad 1 , P/N MLP-9601; or Applied Biosystems, P/N 403083 • 96-well Clear Adhesive Films: Applied Biosystems, P/N 4306311 • Thermal cycler (any Pre-PCR Clean Room thermocycler) • 8-Tube Strips, thin-wall (0.2 mL): Bio-Rad1 , P/N TBS-0201; Strip of 8 caps: Bio-Rad1 , P/N TCS-0801 1 This product was formerly an MJ Research product and is currently available from Bio-Rad.
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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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Page 215 and 216: B2 OLIGO PERFORMANCE chapter 7 | An
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Page 221 and 222: Chapter 8 Troubleshooting
Page 223 and 224: Assay Recommendations 211 Genotypin
Page 225 and 226: chapter 8 | Troubleshooting 213 10.
Page 227 and 228: Problem Likely Cause Solution Faint
Page 229 and 230: Problem Likely Cause Solution Posit
Page 231 and 232: Table 8.2 PROBLEM: Average Sample O
Page 233 and 234: When to Contact Technical Support c
Page 235 and 236: Appendix A Reagents, Equipment, and
Page 237 and 238: Reagents, Equipment, and Consumable
Page 239 and 240: appendix A | Reagents, Equipment, a
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: BEFORE YOU BEGIN appendix C | Low T
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
Page 297 and 298: Appendix D
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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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appendix D | Reagents, Instruments,
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