A simple TAE-based method to generate large insert BAC libraries from plant species

Abstract

Large insert libraries are valuable tools for the positional cloning of genes of interest, physical mapping of chromosomes, comparative genomics, and molecular breeding. There are five types of large DNA insert libraries: cosmid, yeast artificial chromosomes (YACs), bacteriophage P1, bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) libraries. Of these libraries, BAC libraries are the most widely used due to their ease of manipulation, large insert size, and stability. This chapter reports on a simplified method for plant BAC library construction. This method involves isolation and partial digestion of intact nuclei, selection of appropriate size of DNA via pulsed-field gel (PFG) electrophoresis, elution of DNA from agarose gels, ligation of DNA into the BAC vector, electroporation of the ligation mix into Escherichia coli cells, and estimation of insert sizes. The whole process takes 1-3 months depending on the genome size and coverage required. We have used this method to produce BAC libraries from different plant species including sunolgrass (Phalaris coerulescens L.), barley (Hordeum vulgare L.), lupin (Lupinus angustifolias L.) and rye (Secale cereale L.). © 2009 Humana Press, a part of Springer Science+Business Media, LLC.