Rapid extraction of plasmids from Clostridium perfringens

Abstract

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.