Construction and screening of BAC libraries made from Brachypodium genomic DNA

Bacterial artificial chromosome (BAC) libraries are the large DNA insert libraries of choice and valuable tools for the map-based cloning of target quantitative trait loci, physical mapping, molecular cytogenetics and comparative genomics. The protocol reported here is a simplified method used to produce and screen BAC libraries from Brachypodium species and other related grasses. Intact nuclei, containing high molecular weight (HMW) DNA, are isolated and embedded in agarose plugs. The HMW DNA is digested using an appropriate restriction enzyme and size-fractionated using pulsed-field gel electrophoresis. The DNA is isolated by dialysis, ligated into pre-prepared vector and electroporated into competent Escherichia coli cells. A PCR-based method for screening the library is also described. The entire protocol takes at least 6 weeks to complete.