Large-Scale Preparation of Yeast Agarose Plugs to Isolate Yeast Artificial Chromosome DNA

Abstract

The microinjection of DNA directly into the pronuclei of fertilized zygotes is the most extensively used method of gene transfer in the mouse. The injection of very large pieces of DNA, including bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), has become increasingly popular because their size normally accommodates all of the regulatory elements that are needed for a given expression domain to function adequately in an ectopic genomic location. This protocol describes how to prepare large-scale yeast agarose plugs to isolate YAC DNA. The quality of the YAC DNA preparation is the most important consideration in optimizing transgenic mouse production.