CONSTRUCTION OF PLANT BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARIES: AN ILLUSTRATED GUIDE
Daniel G. Peterson1, Jeffrey P. Tomkins2, David A. Frisch2, Rod A. Wing2, and Andrew H. Paterson1, 3
1 Department of Botany, University of Georgia, Athens, GA 30602
2
Clemson University Genomics Institute (CUGI), Clemson University, Clemson, SC 296343
Applied Genetic Technology (AGTEC) Center, Department of Crop and Soil Sciences, and Department of Genetics, University of Georgia, Athens, GA 30602
Corresponding author: Daniel G. Peterson, Plant Genome Mapping Laboratory, University of Georgia, Room 162, Riverbend Research Center, 110 Riverbend Road, Athens, GA 30602, USA, Phone: (706) 583-0167, Fax: (706) 583-0160, e-mail: dgp@arches.uga.edu
Bacterial artificial chromosome (BAC) libraries have become invaluable tools in plant genetic research. However, it is difficult for new practitioners to create plant BAC libraries de novo because published protocols are not particularly detailed, and plant cells possess features that make isolation of clean, high molecular weight DNA troublesome. In this document we present an illustrated, step-by-step protocol for constructing plant BAC libraries. This protocol is sufficiently detailed to be of use to both new and experienced investigators. We hope that by reducing the obstacles to BAC cloning in plants, we will foster new and accelerated progress in plant genomics.
Keywords: bacterial artificial chromosome, BAC, genomics, plant, DNA cloning, physical mapping
INTRODUCTION
CHAPTER 1 Overview
CHAPTER 2 Supplies, equipment, & reagents
VECTOR PREPARATION
CHAPTER 3 Vector isolation
CHAPTER 4 Test ligation
CHAPTER 5 Test transformation
CHAPTER 6 Miniprep & NotI digest
PREPARATION OF INSERT DNA
CHAPTER 7 Isolation of high molecular weight nuclear DNA
CHAPTER 8 DNA analysis
CHAPTER 9 Test restriction digest
CHAPTER 10 Restriction digest
CHAPTER 11 First size selection
CHAPTER 12 Second size selection
CHAPTER 13 Isolation of size-selected DNA from agarose
LIBRARY CONSTRUCTION
CHAPTER 14 Ligation, test transformation, & NotI digest
CHAPTER 15 Mass Transformation
CHAPTER 16 Picking clones
CHAPTER 17 Library replication & storage
APPENDICES
APPENDIX A Using the Gibco BRL Cell-Porator System
APPENDIX B Using the Bio-Rad Model 422 Electroelution System
APPENDIX C Filling 384-well plates using the Genetix QFill2
APPENDIX D The Genetix QBot
APPENDIX E Picking clones using the Genetix QBot
APPENDIX F Library replication using the Genetix QBot