Mapping 250K/500K SNP assay

More documents

Recommendations

Info

88 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual PREPARE DILUTED ALIQUOTS OF PURIFIED SAMPLE Two of the wells on each optical plate must be set up as blanks containing AccuGENE® water only. The 12-channel P20 pipette must be accurate to within ± 5%. To prepare three diluted aliquots of the purified samples: 1. Pour 75 mL of room temperature AccuGENE® water into the solution basin. 2. Using a 12-channel P200 pipette, aliquot 198 µL of water to: A. Each well of optical plates 1, 2 and 3. B. The first four rows of optical plate 4. 3. Using a 12-channel P20 pipette: A. Transfer 2 µL of each purified PCR product from rows A through G of the purified sample plate to the corresponding rows and wells of optical plates 1, 2 and 3 (see Figure 4.5 on page 89). B. Pipette up and down 2 times after each transfer to ensure that all of the product is dispensed. C. Examine the pipette tips and aliquots before and after each dispense to ensure that exactly 2 µL has been transferred. D. Transfer 2 µL of each purified PCR product from row H of the purified sample plate to the corresponding rows and wells of optical plate 4. E. Again, pipette up and down 2 times after each transfer, and examine the pipette tips and aliquots before and after each dispense. The result is a 100-fold dilution. Two of the wells containing water only will serve as blanks. 4. Set a 12-channel P200 pipette to 180 µL. 5. Mix each sample by pipetting up and down 5 to 10 times. Be careful not to scratch the bottom of the plate, or to introduce air bubbles.
Figure 4.5 Loading Optical Plates with Purified Sample chapter 4 | 96-Well Plate Protocol 89 6. Repeat this procedure and prepare three plates of diluted PCR product to test. Be sure to keep two wells as blanks (water only) on each plate. E E E E E E E E E E W W Plate of Purified Samples Optical Plates E = empty W = water only (serve as blanks) Plate of Purified Samples E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E W W W W W W Optical Plate 1 Optical Plate 2 Optical Plate 3 Optical Plate 4
Page 1 and 2:
GeneChip ® Mapping 500K Assay Manu
Page 3 and 4:
Contents CHAPTER 1 Overview 1 ABOUT
Page 5 and 6:
contents v Equipment and Consumable
Page 7 and 8:
contents vii Reagents Required 118
Page 9 and 10:
contents ix CHAPTER 8 Troubleshooti
Page 11 and 12:
contents xi STEP 4: PCR 257 Reagent
Page 13 and 14:
Chapter 1 Overview
Page 15 and 16:
About This Manual This manual is a
Page 17 and 18:
chapter 1 | Overview 5 LD patterns
Page 19 and 20:
Figure 1.1 GeneChip ® Mapping Assa
Page 21 and 22:
chapter 1 | Overview 9 Gilman, B.,
Page 23 and 24:
chapter 1 | Overview 11 23. Schaid,
Page 25 and 26:
chapter 1 | Overview 13 35. Hu, N.,
Page 27 and 28:
chapter 1 | Overview 15 50. Bignell
Page 29 and 30:
chapter 1 | Overview 17 64. Gabriel
Page 31 and 32:
Chapter 2 Laboratory Setup
Page 33 and 34:
Introduction to Laboratory Setup Pl
Page 35 and 36:
Main Lab Pre-PCR Clean Room chapter
Page 37 and 38:
Chapter 3 Genomic DNA General Requi
Page 39 and 40:
Introduction This chapter describes
Page 41 and 42:
Sources of Human Genomic DNA chapte
Page 43 and 44:
chapter 3 | Genomic DNA General Req
Page 45 and 46:
Chapter 4 96-Well Plate Protocol
Page 47 and 48:
ABOUT THIS PROTOCOL 96-Well Plate P
Page 49 and 50: chapter 4 | 96-Well Plate Protocol
Page 51 and 52: PREPARING THE WORK AREA FOR EACH ST
Page 53 and 54: PROGRAM YOUR THERMAL CYCLERS chapte
Page 55 and 56: EQUIPMENT AND CONSUMABLES REQUIRED
Page 57 and 58: ALIQUOTING PREPARED GENOMIC DNA To
Page 61 and 62: Negative Controls chapter 4 | 96-We
Page 63 and 64: Table 4.7 Nsp I Digestion Master Mi
Page 65 and 66: Stage 3: Ligation ABOUT THIS STAGE
Page 67 and 68: REAGENTS REQUIRED chapter 4 | 96-We
Page 71 and 72: DILUTE THE SAMPLES To dilute the sa
Page 73 and 74: Stage 4: PCR ABOUT THIS STAGE chapt
Page 77 and 78: About Controls chapter 4 | 96-Well
Page 79 and 80: B P1 P2 P3 Figure 4.2 Transferring
Page 81 and 82: Table 4.16 PCR Master Mix ADD PCR M
Page 85 and 86: WHAT YOU CAN DO NEXT chapter 4 | 96
Page 91 and 92: POOL THE PCR PRODUCTS chapter 4 | 9
Page 99: chapter 4 | 96-Well Plate Protocol
Page 103 and 104: Process Control Metrics Evaluate th
Page 105 and 106: Table 4.24 PROBLEM: Average Sample
Page 111 and 112: IMPORTANT INFORMATION ABOUT THIS ST
Page 113 and 114: Figure 4.6 Work Area Set Up for Sta
Page 119 and 120: Stage 8: Labeling ABOUT THIS STAGE
Page 127 and 128: LOCATION AND DURATION • Main Lab
Page 129 and 130: Hybridizing Samples Using Heat Bloc
Page 135 and 136: Table 4.37 Hybridization Master Mix
Page 137 and 138: Load the Samples onto Arrays chapte
Page 139 and 140: Figure 4.8 Applying Tough-Spots ®
Page 145 and 146: Chapter 5 Washing, Staining, and Sc
Page 147 and 148: Introduction This chapter contains
Page 149 and 150: Reagent Preparation Wash A: Non-Str
Page 151 and 152:
Experiment and Fluidics Station Set
Page 153 and 154:
chapter 5 | Washing, Staining, and
Page 155 and 156:
Page 157 and 158:
Page 159 and 160:
Page 161 and 162:
HANDLING THE GENECHIP ® PROBE ARRA
Page 163 and 164:
Page 165 and 166:
Chapter 6 Fluidics Station Care and
Page 167 and 168:
Introduction This chapter provides
Page 169 and 170:
chapter 6 | Fluidics Station Care a
Page 171 and 172:
Page 173 and 174:
Page 175 and 176:
Page 177 and 178:
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
Page 225 and 226:
chapter 8 | Troubleshooting 213 10.
Page 227 and 228:
Problem Likely Cause Solution Faint
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 241 and 242:
Page 243 and 244:
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 and 282:
FRAGMENTATION PROCEDURE 1. Pre-heat
Page 283 and 284:
Page 285 and 286:
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 293 and 294:
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 305 and 306:
Page 307 and 308:
Page 309 and 310:
Fragmentation and Labeling appendix
Page 311 and 312:
show all

Mapping 250K/500K SNP assay

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?