Mapping 250K/500K SNP assay

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216 GeneChip ® Mapping 500K Assay Manual Problem Likely Cause Solution Fragmented PCR product is not the correct size PCR product is still visible in 200-1,100 bp size region .CEL file can not be generated GCOS is unable to align grid. Incomplete fragmentation due to underestimated DNA concentration. Failed or incomplete fragmentation due to reduced DNase activity. Unable to place a grid on the .dat file due to the absence of B2 signal. .dat image is dim. Insufficient signal intensity or staining failure. Low SNP call rates Gel images and spectrophotometric quantitation indicate successful PCR reaction. Extremely low call rate Sample hybridization is absent on .cel and .dat images but B2 grid is bright. Incorrect wash buffers used on fluidics station. Over fragmentation of DNA sample due to incorrect dilution of Fragmentation Reagent (DNase I) stock. Ensure spectrophotometer is properly calibrated so only 90 µg of DNA is added to the fragmentation reaction. Check that you have entered the correct activity of DNase into the formula for calculating amount of DNase to add to fragmentation reaction. (See Dilute the Fragmentation Reagent on page 102; amount is the same for high and low throughput.) Ensure fragmentation reagent (DNase I) is kept at –20°C. Do not reuse diluted working stock. Hybridization controls including oligo B2 must be added to hybridization cocktail for grid alignment. Make fresh stain buffers. Prime the fluidics station with the correct buffers prior to running the assay. Incorrect wash buffers will disrupt hybridization of the labeled, fragmented DNA. Use correct concentration of Fragmentation Reagent (DNase I). Check U/µL on the label and check dilution formula (Dilute the Fragmentation Reagent on page 102; amount is the same for high and low throughput). Work quickly and on ice; transfer reaction tubes to preheated thermal cycler (37°C). Mix thoroughly. Labeling reaction suboptimal. Use a new vial of Terminal Dideoxynucleotidyl Transferase. Verify the labeling reagents and repeat labeling.
Problem Likely Cause Solution Positive control has good call rates but samples are lower than expected. chapter 8 | Troubleshooting 217 Genomic DNA not optimal. Ensure DNA samples are of high quality (i.e., run in a 1 to 2% gel and compare to Reference 103 DNA control). Use positive control sample as a reference guide for assay procedures. Prepare master mixes for samples and controls. MDR high, MCR low. Mixed or contaminated genomic DNA sample (page 197). Very low call rates Mixed up Nsp arrays and Sty sample, or vice versa. Use uncontaminated stock of DNA sample. Do Nsp and Sty work on separate days.
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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 177 and 178: chapter 6 | Fluidics Station Care a
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
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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 213 and 214: chapter 7 | Analysis Workflow 201 S
Page 215 and 216: B2 OLIGO PERFORMANCE chapter 7 | An
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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
Page 225 and 226: chapter 8 | Troubleshooting 213 10.
Page 227: Problem Likely Cause Solution Faint
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
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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
Page 277 and 278: 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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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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