Isolation of Plasmids from E. coli by Alkaline Lysis

Abstract

Purification of plasmid DNA from Escherichia coli using alkaline lysis (1,2) is based on the differential denaturation of chromosomal and plasmid DNA in order to separate the two. Bacteria are lysed with a solution containing sodium dodecyl sulfate (SDS) and sodium hydroxide. During this step, chromosomal as well as plasmid DNA are denatured. Subsequent neutralization with potassium acetate allows only the covalently closed plasmid DNA to reanneal and to stay solubilized. Most of the chromosomal DNA and proteins precipitate in a complex formed with potassium and SDS, which is removed by centrifugation. The plasmid DNA is concentrated from the supernatant by ethanol precipitation. Using this procedure, 2–5 μg of DNA can be obtained from a 1.5-mL culture of E. coli containing a pBR322-derived plasmid, and three- to five-fold higher yields can be expected from pUC-derived plasmids (3).