Mapping 250K/500K SNP assay

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298 GeneChip ® Mapping 500K Assay Manual Table D.6 Reagents, Equipment and Supplies Not Supplied by Affymetrix Assay Step Reagents Equipment Target Hybridization 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 0.5 M 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 Wash and Staining 20X SSPE: BioWhittaker Molecular Applications/ Cambrex, P/N 51214 Denhardt’s Solution, 50X concentrate: Sigma, P/N D2532 10% Tween-20: Pierce, P/N 28320 (Surfact-Amps ® ) Molecular Biology Grade Water: BioWhittaker Molecular Applications/Cambrex, P/N 51200 SAPE (Streptavidin, R-phycoerythrin conjugate): Invitrogen, P/N S866, 1 mg/mL* Ab (Biotinylated Anti-Streptividin): Vector Laboratories, P/N BA-0500, 0.5mg;* reconstitute according to product instructions Distilled water: Invitrogen Life Technologies, P/N 15230147 Bleach (5.25% Sodium Hypochlorite): VWR Scientific, P/N 21899-504 (or equivalent) GeneChip ® Hybridization Oven 640 GeneChip ® Fluidics Station 450 Scanning GeneChip ® Scanner 3000 7G * 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. † dNTPs from Invitrogen (P/N R72501) have been tested on a limited basis with similar results. You should test in your own lab prior to full scale production.
appendix D | Reagents, Instruments, and Supplies Required for Low Throughput Protocol 299 ‡ The PCR process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-LaRoche Ltd (“Roche”). A license to use the PCR process for certain research and development activities accompanies the purchase of certain reagents from licensed suppliers when used in conjunction with an authorized thermal cycler. If you are using an MJ Research thermal cycler, your thermal cycler may not be an authorized thermal cycler. You should obtain authorization from Roche or ABI (see PCR licensing information in the MJ Research User Manual), if you are not already licensed. For information about obtaining a license contact The Director of Licensing at Applied Biosystems, 850 Lincoln Center Drive, Foster City, CA 94404 or the Licensing Department, Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, CA 94501. ** In 2004 MJ GeneWorks, Inc. and its subsidiary MJ Research, Inc. were purchased by Bio-Rad Laboratories, Inc. This model is no longer available. Bio-Rad has indicated that the DNA Engine Tetrad 2 gives similar performance with the same progams. Affymetrix has not tested the newer version. †† ® ® Clontech also supports the Millipore MultiScreen , Promega Vac-Man 96, and BioRad Aurum vacuum manifolds for use with the Clontech Clean-Up Plates. ‡‡ The Clontech protocol requires ~600 mb vacuum. If your lab does not have an internally regulated vacuum source, this vacuum regulator is strongly sug- gested.
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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
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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
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
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: Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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