COMPOSITIONS AND METHODS FOR RAPID ISOLATION OF PLASMID DNA

摘要

A composition for isolating plasmid DNA from a bacterial culture is disclosed,the composition containing at least about 3 M magnesium chloride and about 10% (w/v) polyethylene glycol. A method of differentially precipitating plasmidDNA from a bacterial lysate is also disclosed, involving the steps of lysingbacterial cells to obtain a bacterial lysate; mixing magnesium chloride andpolyethylene chloride with the lysate to form a mixture containing at leastabout 1 M magnesium chloride and about 3.3 % (w/v) polyethylene glycol,thereby precipitating cellular debris, chromosomal DNA, cellular RNA, andproteins; separating and removing the precipitated material to form a clearedlysate; mixing magnesium chloride and polyethylene glycol with the clearedlysate to form a mixture containing at least about 1.5 M magnesium chlorideand about 5 % (w/v) polyethylene chloride, thereby precipitating plasmid DNA;and separating the precipitated plasmid DNA from the mixture, therebyobtaining isolated plasmid DNA. Another composition according to the inventioncontains an enzyme for digestion of cellular RNA, a stabilizer for protectingplasmid DNA from nuclease digestion, and a precipitating agent forprecipitating plasmid DNA. Another method of isolating plasmid DNA from abacterial culture comprises simultaneously digesting cellular RNA in aclarified lysate with an enzyme, protecting plasmid DNA from nucleasedigestion, and precipitating plasmid DNA.