Purification of Yeast Artificial Chromosome DNA for Microinjection Using a Two-Gel Electrophoresis Procedure

This protocol describes a method to purify yeast artificial chromosome (YAC) DNA for microinjection. YAC DNA solutions cannot be concentrated by standard DNA precipitation methods and resuspended into smaller volumes. Attempts to precipitate YAC DNA solutions will, unavoidably, break and destroy the DNA molecules. Therefore YAC DNA must always be kept in a solution that maintains the integrity of the YAC DNA molecules. In the method described here, YAC DNA is extracted from pulsed-field gel electrophoresis (PFGE) agarose plugs by using two gel electrophoresis steps. The first gel electrophoresis step is the PFGE itself. The region of agarose containing the YAC DNA of interest is excised and run on a standard electrophoresis gel setup at a 90° angle to the PFGE run. The YAC DNA migrates out of the agarose slice and enters into a thicker low-melting agarose gel, thereby promoting compaction of YAC DNA molecules. The aim of this method is to convert a slice of agarose into a cube of agarose of smaller volume. The volume of this cube will determine the final concentration of the YAC DNA precipitation.