Mapping 250K/500K SNP assay

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220 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual Table 8.2 PROBLEM: Average Sample OD is Less Than 0.5 (2.5 µg/µL) Troubleshooting Possible Problems with the Elution or OD Readings – possible causes include: The purified PCR product was eluted in a volume greater than 45 µL. The purified PCR product was not mixed adequately before making the 1:100 dilution. The diluted PCR product was not mixed adequately before taking the OD reading. The water blank reading was not subtracted from each sample OD reading. The spectrophotometer plate reader may require calibration. Pipettes may require calibration. There may be air bubbles or dust in the OD plate. There may be defects in the plastic of the plate. The settings on the spectrophotometer plate reader or the software may be incorrect. OD calculations may be incorrect and should be checked. Reliance on any single OD reading may give an outlier result. You should make three independent dilutions and take three independent OD readings per dilution. Table 8.3 PROBLEM: OD260/OD280 ratio is not between 1.8 and 2.0 Possible causes include: The PCR product may be not be sufficiently purified. Be sure to perform three water washes and check to be sure the vacuum manifold is working properly. An error may have been made while taking the OD readings. Table 8.4 PROBLEM: The OD320 measurement is significantly larger than zero (0 ± 0.005) Possible causes include: Precipitate may be present in the eluted samples. Be sure to add diluted EDTA to PCR products before purification. There may be defects in the OD plate. Air bubbles in the OD plate or in solutions.
When to Contact Technical Support chapter 8 | Troubleshooting 221 Under any of the following conditions, unplug the instrument from the power source and contact Affymetrix Technical Support: • when the power cord is damaged or frayed; • if any liquid has penetrated the instrument; • if, after service or calibration, the instrument does not perform to the specifications stated in Instrument Specifications on page 178. If the instrument must be returned for repair, call Affymetrix Technical Support. Make sure you have the model and serial number. Affymetrix, Inc. 3420 Central Expressway Santa Clara, CA 95051 USA E-mail: support@affymetrix.com Tel: 1-888-362-2447 (1-888-DNA-CHIP) Fax: 1-408-731-5441 Affymetrix UK Ltd Voyager, Mercury Park, Wycombe Lane, Wooburn Green, High Wycombe HP10 0HH United Kingdom E-mail: supporteurope@affymetrix.com UK and Others Tel: +44 (0) 1628 552550 France Tel: 0800919505 Germany Tel: 01803001334 Fax: +44 (0) 1628 552585
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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
Page 181 and 182: Figure 6.8 Troubleshooting decision
Page 183 and 184: PROBLEMS AND SOLUTIONS Table 6.3 Co
Page 185 and 186: Table 6.3 (Continued) Common error
Page 187 and 188: Table 6.4 (Continued) Common Error
Page 189 and 190: Table 6.5 (Continued) Other Problem
Page 191 and 192: Chapter 7 Analysis Workflow
Page 193 and 194: Introduction Software Requirements
Page 195 and 196: Figure 7.2 GTYPE main window with t
Page 197 and 198: Figure 7.3 Dynamic Model Mapping Al
Page 199 and 200: Table 7.1 Dynamic Model Mapping Alg
Page 201 and 202: FILE SETS chapter 7 | Analysis Work
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Page 205 and 206: NETAFFX SNP ANNOTATION chapter 7 |
Page 207 and 208: CALL RATE chapter 7 | Analysis Work
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Page 211 and 212: chapter 7 | Analysis Workflow 199 F
Page 213 and 214: chapter 7 | Analysis Workflow 201 S
Page 215 and 216: B2 OLIGO PERFORMANCE chapter 7 | An
Page 217 and 218: chapter 7 | Analysis Workflow 205 3
Page 219 and 220: chapter 7 | Analysis Workflow 207 W
Page 221 and 222: Chapter 8 Troubleshooting
Page 223 and 224: Assay Recommendations 211 Genotypin
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Page 227 and 228: Problem Likely Cause Solution Faint
Page 229 and 230: Problem Likely Cause Solution Posit
Page 231: Table 8.2 PROBLEM: Average Sample O
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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
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Page 279 and 280: STEP 7: Fragmentation REAGENTS AND
Page 281 and 282: FRAGMENTATION PROCEDURE 1. Pre-heat
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appendix C | Low Throughput Protoco
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LABELING PROCEDURE MAIN LAB appendi
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STEP 9: Target Hybridization REAGEN
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HYBRIDIZATION PROCEDURE appendix C
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Appendix D Reagents, Instruments, a
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Appendix D
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appendix D | Reagents, Instruments,
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* Adaptor, Nsp 5' ATTATGAGCACGACAGA
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Table D.4 Reagents Supplied by Affy
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Fragmentation and Labeling appendix
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Mapping 250K/500K SNP assay

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