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 29634

3 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

ABSTRACT

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

TABLE OF CONTENTS

ABSTRACT


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


GLOSSARY


ACKNOWLEDGEMENTS


REFERENCES