Method for obtaining more accurate covalently closed circular plasmid to chromosome ratios from bacterial lysates by dye buoyant density centrifugation

Abstract

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This methpd should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid host combinations.