A simple method for the qualitative demonstration of large and small plasmid deoxyribonucleic acids in Gram-positive and Gram-negative bacteria

Abstract

The method of Currier and Nester ( Anal. Biochem. 76, 431–441, 1976) was modified so that large (molecular weights greater than 4 × 10 6) and small (molecular weights less than 4 × 10 6) covalently closed circular (plasmid) deoxyribonucleic acid (DNA) molecules could be demonstrated in agarose gels with little or no interference by chromosomal DNAs and linear and open circular forms of plasmid DNAs. The major modifications consisted of treating cells with a lytic enzyme in the presence of at least 10% (w/v) sucrose (essential for demonstrating large plasmids) and denaturing chromosomal DNA by boiling lysates for 15 min and quenching them in ice water instead of treating them at high pH. A minor modification consisted of boiling cell lysates in the presence of 200 μg/ml ethidium bromide. The method described allowed the detection of plasmid DNAs in a large number (24 or more) of overnight 10-ml cultures within a reasonable time (2 working days). The modification as presented is applicable to both Gram-positive and Gram-negative bacteria.