Generating subclones from large-insert genomic clones

This unit provides a simple protocol for generating a partial-digest sublibrary of yeast DNA containing a YAC of interest. The starting material is high-molecular-weight chromosomal DNA embedded in agarose plugs. Many genome equivalents of these YAC subclones in bacteriophage or cosmid vectors can be plated and screened by hybridization with total human DNA to identify clones that originate from the human portion of the YAC. The human-positive clones can be picked into 96-well microtiter plates for spotting on membranes. Once stored in ordered arrays, the YAC subclones can be constructed into contigs using an end-probe or Alu-PCR hybridization strategy, or a gel fingerprinting technique. This unit provides a simple protocol for generating a partial-digest sublibrary of yeast DNA containing a YAC of interest.