Mapping 250K/500K SNP assay

More documents

Recommendations

Info

106 GeneChip ® Mapping 500K Assay Manual CHECK THE FRAGMENTATION REACTION To ensure that fragmentation was successful: 1. When the 500K Fragment program is finished: A. Remove the plate from the thermal cycler. B. Spin down the plate at 2000 rpm for 30 sec, and place in a cooling chamber on ice. 2. Dilute 4 µL of each fragmented PCR product with 4 µL gel loading dye. 3. Run on 4% TBE gel with the BioNexus All Purpose Hi-Lo ladder at 120V for 30 minutes to 1 hour. 4. Inspect the gel and compare it against the example shown in Figure 4.7 below. Figure 4.7 Typical example of fragmented PCR products run on 4% TBE agarose gel at 120V for 30 minutes to 1 hour. Average fragment size is < 180 bp.
Stage 8: Labeling ABOUT THIS STAGE chapter 4 | 96-Well Plate Protocol 107 During this stage, the fragmented samples will be labeled using the GeneChip® DNA Labeling Reagent. You will prepare the Labeling Master Mix, add the mix to each sample, place the samples onto a thermal cycler and run the 500K Label program. LOCATION AND DURATION • Main Lab • Hands-on time: 30 minutes • 500K Label thermal cycler program time: 4.25 hours INPUT REQUIRED FROM PREVIOUS STAGE The input required from Stage 7: Fragmentation is: Quantity Item 1 Plate of fragmented DNA EQUIPMENT AND CONSUMABLES REQUIRED The following equipment and consumables are required for this stage. Refer to Appendix A, Reagents, Equipment, and Consumables Required for 96-Well Plate Protocol for vendor and part number information. ** Use only the PCR plate, adhesive film and thermal cyclers listed in Table 4.1 on page 40.
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 59 and 60:
chapter 4 | 96-Well Plate Protocol
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 69 and 70: chapter 4 | 96-Well Plate Protocol
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 87 and 88: EQUIPMENT AND CONSUMABLES REQUIRED
Page 91 and 92: POOL THE PCR PRODUCTS chapter 4 | 9
Page 101 and 102: Figure 4.5 Loading Optical Plates w
Page 103 and 104: Process Control Metrics Evaluate th
Page 105 and 106: Table 4.24 PROBLEM: Average Sample
Page 109 and 110: EQUIPMENT AND CONSUMABLES REQUIRED
Page 111 and 112: IMPORTANT INFORMATION ABOUT THIS ST
Page 113 and 114: Figure 4.6 Work Area Set Up for Sta
Page 117: WHAT YOU CAN DO NEXT chapter 4 | 96
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 161 and 162: HANDLING THE GENECHIP ® PROBE ARRA
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?