Mapping 250K/500K SNP assay

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Pre-PCR Clean Room PCR Staging Room 22 GeneChip ® Mapping 500K Assay Manual • Use dedicated equipment (e.g., pipets, tips, thermocyclers, etc.) for pre-PCR stages. • Print separate copies of the protocol for each room. In addition, the following areas should be used when performing this assay: pre-PCR Clean Room, PCR Staging Room, and Main Lab. A summary of the areas is shown in Figure 2.1. Once you enter the Main Lab, do not return to the Pre-PCR Room or the PCR Staging Room until you have showered and changed into freshly laundered clothing. The Pre-PCR Clean Room (or dedicated area such as a biosafety hood) should be free of DNA template and PCR amplicons. The master stocks of PCR primer and adaptor should be stored here, with aliquots taken for use in the PCR Staging Room (or area). Reagent preparation tasks, such as preparing master mixes, should be done in this room. The use of gowns, booties, and gloves is strongly recommended to prevent PCR carryover and to minimize the risk of trace levels of contaminants being brought into the Pre-PCR Clean Area. This room should contain dedicated pipets, tips, vortex, etc. Refer to Appendix A or Appendix D as appropriate for more details. The PCR Staging Room (or Area) is a low copy template lab, which should be free from any PCR product (amplicons). It is the area where non-amplified template (genomic DNA) should be handled. The digestion and ligation reactions should be conducted in this area. The PCR reactions should be prepared in this area. The use of gowns, booties, and gloves is recommended to prevent PCR carryover. Each room should contain dedicated equipment such as thermocyclers, microfuges, pipets, tips, etc.
Main Lab Pre-PCR Clean Room chapter 2 | Laboratory Setup 23 The Main Lab has airborne contamination with PCR product and template. After entering the main lab it is inadvisable to re-enter the Pre-PCR Clean Area or the PCR Staging Area without first showering and changing into freshly laundered clothes. Assay Steps: Step 1: Reagent Preparation PCR Staging Room Assay Steps: Step 2: Enzyme Digestion Step 3: Ligation Step 4: PCR (set up only) Main Lab Assay Steps: Step 4: PCR Thermal Cycling Step 5: PCR Clean up Step 6: Fragmentation Step 7: Labeling Step 8: Hybridization Step 9: Wash and Stain Template (Genomic DNA) PCR Product Figure 2.1 A summary of rooms required to perform the GeneChip ® Mapping 500K Assay
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: Introduction to Laboratory Setup Pl
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 75 and 76: REAGENTS REQUIRED chapter 4 | 96-We
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
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WHAT YOU CAN DO NEXT chapter 4 | 96
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EQUIPMENT AND CONSUMABLES REQUIRED
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chapter 4 | 96-Well Plate Protocol
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POOL THE PCR PRODUCTS chapter 4 | 9
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REAGENTS REQUIRED chapter 4 | 96-We
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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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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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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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