Mapping 250K/500K SNP assay

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Before You Begin MASTER MIX PREPARATION 38 GeneChip ® Mapping 500K Assay Manual Carefully follow each master mix recipe. Use pipettes that have been calibrated to ± 5%. When molecular biology-grade water is specified, be sure to use the AccuGENE® water listed in Appendix A, Reagents, Equipment, and Consumables Required for 96-Well Plate Protocol. Using in-house ddH2O or other water can negatively affect your results. The enzymatic reaction in Stage 7: Fragmentation is particularly sensitive to pH and metal ion contamination. If you run out of master mix during any of these procedures, a volume error has been made or the pipettes are not accurate. We recommend that you stop and repeat the experiment. REAGENT HANDLING AND STORAGE Follow these guidelines for reagent handling and storage. • When working on the bench top, keep all reagents (except enzymes) on ice in a cooling chamber that has been chilled to 4 °C. • Always leave enzymes at –20 °C until immediately prior to adding them to master mixes. When removed from the freezer, immediately place in a cooler that has been chilled to –20 °C and placed on ice. • Store the reagents used for the restriction digestion, ligation and PCR steps in the Pre-PCR Clean Area. • Do not re-enter the Pre-PCR Clean Area after entering the PCR- Staging Room or the Main Lab. Aliquot each of the reagents in the Pre-PCR Clean Area before starting the rest of the experiment. • Dedicate one cooler for the Pre-PCR Clean Area and one for the Main Lab (post-PCR). • When performing the steps for Stages 2 through 8: - Leave all of the tubes on ice or in a cooling chamber on ice. - Keep all plates in a cooling chamber on ice. • Consult the appropriate MSDS for reagent storage and handling requirements.
PREPARING THE WORK AREA FOR EACH STAGE Reagent (thawed, vortexed and spun down) Eppendorf tube labeled and ready for master mix preparation Figure 4.1 Example of Work Area Preparation chapter 4 | 96-Well Plate Protocol 39 Many of the stages in the Mapping 500K 96-well plate protocol must be performed rapidly and on ice to carefully control enzyme activity and temperature transitions. Therefore, we recommend that you gather and set up all of the equipment, consumables and reagents (except for the enzymes) prior to beginning each stage. Below is an illustration of the setup for Stage 7: Fragmentation. Pipettes and tips are not shown. Cooling chamber, chilled to 4 °C Two strips of tubes, each row labeled and ready for additions Plate of samples from previous stage Cooler, chilled to –20 °C Have pipettes and pipette tips ready as well. Begin each stage with full racks of pipette tips.
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: chapter 4 | 96-Well Plate Protocol
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 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 95 and 96: WHAT YOU CAN DO NEXT chapter 4 | 96
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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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WHAT YOU CAN DO NEXT chapter 4 | 96
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EQUIPMENT AND CONSUMABLES REQUIRED
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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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chapter 4 | 96-Well Plate Protocol
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Stage 8: Labeling ABOUT THIS STAGE
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REAGENTS REQUIRED chapter 4 | 96-We
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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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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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