Large genomic DNA insert-containing libraries are required as critical tools for physical
mapping, positional cloning, and genome sequencing of complex genomes. The bacterial artificial
chromosome (BAC) cloning system has become a dominant system over others to clone
large genomic DNA inserts. As the costs of positional cloning, physical mapping, and genome
sequencing continuously decrease, there is an increasing demand for high-quality deepcoverage
large insert BAC libraries. In our laboratory, we have constructed many high-quality
deep-coverage large insert BAC libraries including arabidopsis, manocot and dicot crop plants,
and plant pathogens. Here, we present the protocol used in our laboratory to construct BAC
libraries.