CytoScan™ Assay USER GUIDE Manual Workflow Catalog Numbers 901835, 901834, 901859, 901860 Publication Number 703038 Revision 7 For Research Use Only. Not for use in diagnostic procedures. The information in this guide is subject to change without notice. DISCLAIMER TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses. Corporate entity Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288 TRADEMARKS All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Microsoft, and Excel are either registered trademarks or trademarks of Microsoft Corporation in the United States and/or other countries. QIAGEN, Gentra and Puregene are registered trademarks of QIAGEN. Titanium is a trademark of Clontech Laboratories, Inc. Tough-Spots is a registered trademark of Diversified Biotech. ©2011, 2014, 2017 Thermo Fisher Scientific Inc. All rights reserved. Contents CHAPTER 1 Before you begin. 6 Introduction . 6 Assay warnings and precautions . 7 Genomic DNA general requirements and recommendations . 9 Sources of human genomic DNA . 10 Genomic DNA extraction/purification methods . 10 RNase treatment . 10 Controls . 10 Data analysis . 11 CHAPTER 2 CytoScan™ Assay procedure . 12 Temperature definitions . 12 Section 1: Prepare gDNA plate . 13 Dilute stock gDNA to working concentration . 13 Aliquot prepared gDNA into assay plate . 13 Section 2: Restriction enzyme digestion . 15 Prepare reagents, equipment, and consumables . 15 Prepare reagents . 15 Load samples on thermal cycler . 17 Section 3: Ligation . 18 Section 4: PCR . 22 Dilute ligated samples . 22 Run PCR . 23 Check PCR reaction . 27 Section 5: PCR product purification . 28 Prepare Purification Wash Buffer . 28 Prepare PCR products . 28 Section 6: Quantitation . 32 Prepare reagents, equipment, and consumables . 32 Microplate spectrophotometer . 32 Section 7: Fragmentation . 34 Precautions . 34 CytoScan™ Assay Manual Workflow User Guide 3 Contents Preparation . 34 Fragmentation . 36 Section 8: Labeling . 39 Preparation . 39 Labeling . 40 Section 9: Target hybridization via AGCC . 42 Important information about this stage . 42 Prepare the equipment . 42 Prepare the arrays and create a Batch Registration file . 42 Prepare the reagents and consumables . 44 Prepare the Hybridization Master Mix . 46 Add Hybridization Master Mix and denature . 46 Load the samples onto arrays . 47 Section 10: Washing, staining, and scanning arrays . 48 Prime the fluidics station . 48 Washing and staining arrays. 49 Scanning arrays . 50 Adding arrays during an AutoLoader run . 51 Shutting down the fluidics station . 52 Section 11: In-process quality control . 53 Diluting the TrackIt™ Cyan/Orange Loading Buffer . 53 PCR product . 53 Fragmented product . 54 Section 12: Array quality control threshold . 56 APPENDIX A Workflow and practices. 57 Workflow . 57 Specific laboratory practices . 59 Seals . 59 Vortex . 59 Centrifuge . 60 PCR practices . 60 Samples . 60 PCR product purification . 60 APPENDIX B Alternative assay steps . 61 Alternative preparation of Fragmentation Master Mix . ..