Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation.
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 method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.
D002499:Centrifugation, Density Gradient; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004270:DNA, Circular / Q000302:isolation & purification*; D004926:Escherichia coli; D005111:Extrachromosomal Inheritance; D008722:Methods; D010957:Plasmids; D011513:Proteus mirabilis
D004269:DNA, Bacterial; D004270:DNA, Circular