A rapid alkaline extraction procedure for screening recombinant plasmid DNA

TLDR

The paper describes a procedure for extracting plasmid DNA from bacterial cells. The method uses selective alkaline denaturation of chromosomal DNA, leaving covalently closed circular plasmid DNA intact, with pH control achieved without a meter and neutralization causing chromosomal DNA to clot while plasmid DNA remains in the supernatant. The method allows rapid screening of 100+ clones per day, producing plasmid DNA pure enough for restriction digestion and successfully extracting both large and small plasmids.

Abstract

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.