Mapping 250K/500K SNP assay

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270 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual As the concentration of stock Fragmentation Reagent (U/µL) may vary from lot to lot, it is essential to check the concentration before conducting the dilution. Do calculations prior to diluting sample and Fragmentation Reagent. To ensure uniform and reproducible fragmentation, completely pipet the viscous reagent from the stock vial into the diluted enzyme mix. Thorougly mix the diluted enzyme before aliquoting to the DNA samples. The Fragmentation Reagent must be added to the fragmentation mix quickly and ON ICE to minimize enzyme activity prior to placing the samples on the thermal cycler. 4. Dilute the stock of Fragmentation Reagent to 0.05 U/µL as follows: A. Place the water (Molecular Biology Grade), Fragmentation Buffer, and Fragementation Reagent on ice. B. Combine the reagents ON ICE in the order shown in Table C.10. C. Vortex at medium speed for 2 seconds. Two examples of dilution are listed below for two different concentrations of Fragmentation Reagent.
appendix C | Low Throughput Protocol 271 Table C.10 Diluting the Fragmentation Reagent – Combine Reagents in the Order Shown Reagent 2 units/µL 3 units/µL H 2O, Molecular Biology Grade 105 µL 106 µL 10X Fragmentation Buffer 12 µL 12 µL Fragmentation Reagent* 3 µL 2 µL Total 120 µL 120 µL * Add the Fragmentation Reagent last, after allowing the water and buffer to cool on ice. If the concentration on your tube is not shown in the table above, use the formula provided in Step 3. 5. Quickly and ON ICE, divide the diluted Fragmentation Reagent into 8 or 12 microtube strips. 6. Add 5 µL of diluted Fragmentation Reagent (0.05 U/µL) with an 8- or 12-channel pipette to the fragmentation plate containing Fragmentation Mix ON ICE. Pipet up and down several times to mix. Be sure to change tips between samples. The total volume for each sample is listed below. Table C.11 Reagent Volume/Sample Fragmentation Mix 50 µL Diluted Fragmentation Reagent (0.05 U/µL) 5 µL Total 55 µL For 90 µg of purified PCR product, a total of 0.25 U of Fragmentation Reagent is needed in a final reaction volume of 55 µL. 7. Cover the fragmentation plate with a plate cover and seal tightly.
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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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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
Page 277 and 278: appendix C | Low Throughput Protoco
Page 279 and 280: STEP 7: Fragmentation REAGENTS AND
Page 281: 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
Page 299 and 300: appendix D | Reagents, Instruments,
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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