A method for the preparation of high molecular weight yeast DNA.

A method is described for the isolation of high molecular weight DNA in solution using the principles that have allowed electrophoresis of chromosome-sized DNA in pulse field gradient electrophoresis. Stationary phase yeast cells are converted to spheroplasts by the action of zymolyase in 1 M sorbitol. In the presence of EDTA and sodium lauroyl sarcosinate, proteins are digested with proteinase K. DNA is extracted with phenol and chloroform, and high molecular weight DNA is collected by ethanol precipitation. RNA is removed by RNase digestion of the redissolved pellet, and RNase is removed by chloroform extraction followed by a second ethanol precipitation. The method is rapid and gives a high yield of DNA that is readily digestible by restriction endonucleases.