Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants

Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.