Mapping 250K/500K SNP assay

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102 GeneChip ® <strong>Mapping</strong> <strong>500K</strong> Assay Manual PREPARE THE SAMPLES FOR FRAGMENTATION Add Fragmentation Buffer to Samples All of the additions in this procedure must be performed on ice. To prepare the samples for Fragmentation: 1. Aliquot 50 µL of 10X Fragmentation Buffer to each tube of the 12-tube strip labeled Buffer. 2. Using a 12-channel P20 pipette, add 5 µL of Fragmentation Buffer to each sample in the 96-well reaction plate. Check your pipette tips each time to ensure that all of the buffer has been dispensed. The total volume in each well is now 50 µL. Dilute the Fragmentation Reagent The concentration of stock Fragmentation Reagent (U/µL) may vary from lot-to-lot. Therefore, read the label on the tube and record the stock concentration before diluting this reagent. Use the formula provided to accurately calculate the dilution required. To dilute the Fragmentation Reagent: 1. Read the Fragmentation Reagent tube label and record the concentration.
chapter 4 | 96-Well Plate Protocol 103 Table 4.30 Dilution Recipes for Fragmentation Reagent Concentrations of 2 and 3 U/µL Reagent 2. If the concentration is 2 or 3 U/µL, dilute the Fragmentation Reagent using the volumes show in Table 4.30 above. 3. If the concentration is not 2 or 3 U/µL, use the formula below to calculate the dilution required to bring the reagent to a final concentration of 0.05 U/µL. 4. Dilute the Fragmentation Reagent to 0.05 U/µL as follows using the recipes in Table 4.30 or the dilution formula calculation: A. To the 1.5 mL Eppendorf tube on ice: Fragmentation Reagent Concentration 2 U/µL 3U/µL AccuGENE® water 525 µL 530 µL Fragmentation Buffer 60 µL 60 µL Fragmentation Reagent 15 µL 10 µL Total (enough for 96 samples) Formula Y = 0.05 U/µL * 600 µL X U/µL Where: 600 µL 600 µL Y = number of µL of stock Fragmentation Reagent X = number of U of stock Fragmentation Reagent per µL (per label on tube) 0.05 U/µL = final concentration of Fragmentation Reagent 600 µL = final volume of diluted Fragmentation Reagent (enough for 96 reactions) 1) Add the AccuGENE® water and Fragmentation Buffer.
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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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chapter 4 | 96-Well Plate Protocol
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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: 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 161 and 162: 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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Table 8.2 PROBLEM: Average Sample O
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When to Contact Technical Support c
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Appendix A Reagents, Equipment, and
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Reagents, Equipment, and Consumable
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appendix A | Reagents, Equipment, a
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Consumables Required GENECHIP ® AR
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Supplier Contact List Table A.10 Su
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Appendix B Thermal Cycler Programs
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ABOUT THIS APPENDIX 500K DIGEST 500
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500K FRAGMENT 500K LABEL 500K HYB a
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Appendix C Low Throughput Protocol
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Introduction The Affymetrix GeneChi
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BEFORE YOU BEGIN appendix C | Low T
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STEP 2: Restriction Enzyme Digestio
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DIGESTION PROCEDURE PRE-PCR CLEAN A
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STEP 3: Ligation REAGENTS AND EQUIP
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PCR STAGING AREA appendix C | Low T
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STEP 4: PCR REAGENTS AND EQUIPMENT
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PCR PROCEDURE PRE-PCR CLEAN ROOM ap
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MAIN LAB appendix C | Low Throughpu
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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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