Mapping 250K/500K SNP assay

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214 GeneChip ® Mapping 500K Assay Manual Troubleshooting Guide for the GeneChip ® Mapping 500K Assay Problem Likely Cause Solution Faint/absent bands on PCR gel Both samples & positive control affected. Problem with master mixes or individual reagents. Ensure all reagents added to master mixes and enzymes are stored at –20°C. Work quickly with enzymes and return to –20°C directly after use to prevent loss of activity. Failed restriction digest. Use restriction enzyme to digest a known good DNA sample. Run gel to confirm restriction enzyme activity. Failed adaptor ligation reaction. Confirm enzyme activity. Reduced adaptor ligation efficiency due to adaptor self-ligation, DNA religation. Ligase buffer contains ATP and should be defrosted/ held at 4°C. Mix ligase buffer thoroughly before use to ensure precipitate is re-suspended. Avoid multiple freeze-thaw cycles. Try a fresh tube of buffer. To prevent self-ligation of adaptor work rapidly and add DNA ligase last. Failed PCR reaction. Check PCR reagents. Take care with preparation of master mixes and ensure accurate pipetting and thorough mixing. Reduced PCR reaction yield – non optimal PCR conditions. Ligation mix not diluted prior to PCR reaction. Incorrect concentration of nucleotides. Used Nsp adaptor for Sty digest, or vice versa. Use a validated thermal cycler, check PCR programs. Use recommended thin walled reaction tubes. Thoroughly mix PCR reaction. Ligation mixture diluted 1:4 with molecular biology grade water to remove potential inhibitors and maintain optimal pH and salt concentration. Check dNTP stock concentration and vendor. Repeat Ligation step with correct adaptors.
Problem Likely Cause Solution Faint/absent bands on PCR gel (continued) Samples affected (but positive controls OK). Low PCR yield Gel image shows PCR product but following purification spectrophotometer measurements at 260nm indicate low PCR yield. chapter 8 | Troubleshooting 215 Non-optimal reaction conditions. Use master mixes and include a positive control to eliminate reagents and assay problems as detailed above. Insufficient starting material. 250 ng genomic DNA should be used. Confirm concentration using calibrated spectrophotometer. Sample DNA contains enzymatic or chemical inhibitors. Ensure genomic DNA is purified and diluted in Low EDTA (0.1mm) TE buffer. Use recommended procedure to ethanol precipitate genomic DNA to remove inhibitors. Degraded sample DNA. Confirm quality of genomic DNA sample. Sufficient PCR product is present but spectrophotometer is out of calibration. PCR product is lost due to reduced temperature. Pipette used for quantitation not calibrated. White precipitate is seen after the PCR reaction Calibrate spectrophotometer. Proceed with assay. All purification steps must be carried out at 25 to 35°C. Calibrate Pipette. Proceed with assay. Precipitation from assay reagents. Add 0.1 M EDTA, as per protocol, before PCR product purification. This precipitate should no longer be present after purification.
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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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Page 175 and 176: chapter 6 | Fluidics Station Care a
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Page 179 and 180: 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
Page 203 and 204: chapter 7 | Analysis Workflow 191 3
Page 205 and 206: NETAFFX SNP ANNOTATION chapter 7 |
Page 207 and 208: CALL RATE chapter 7 | Analysis Work
Page 209 and 210: chapter 7 | Analysis Workflow 197 G
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 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 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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appendix C | Low Throughput Protoco
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STEP 7: Fragmentation REAGENTS AND
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FRAGMENTATION PROCEDURE 1. Pre-heat
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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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