Purification of Yeast Artificial Chromosome DNA for Microinjection Using Pulsed-Field Gel Electrophoresis and Ultrafiltration

Abstract

This protocol describes a method for concentrating yeast artificial chromosome (YAC) DNA preparations following separation of chromosomes by pulsed-field gel electrophoresis (PFGE). The agarose slice containing the YAC DNA of interest is melted and digested. The resulting YAC DNA-containing solution is concentrated using ultrafiltration units with a 30,000-mwt cutoff. The DNA is retained in the upper container, where it concentrates sixfold to sevenfold. Ideally, YAC DNA preparations at 10–60 ng/µL can be obtained. However, great care must be taken to recover the YAC DNA molecules that sometimes irreversibly attach to the filter membrane. This procedure can also be used to concentrate BAC DNA from PFGE gel slices of BAC DNA-containing agarose exactly as described for YACs.