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 plasmid DNA from a bacterial lysate is also disclosed, involving the steps of lysing bacterial cells to obtain a bacterial lysate; mixing magnesium chloride and polyethylene chloride with the lysate to form a mixture containing at least about 1 M magnesium chloride and about 3.3 % (w/v) polyethylene glycol, thereby precipitating cellular debris, chromosomal DNA, cellular RNA, and proteins; separating and removing the precipitated material to form a cleared lysate; mixing magnesium chloride and polyethylene glycol with the cleared lysate to form a mixture containing at least about 1.5 M magnesium chloride and about 5 % (w/v) polyethylene chloride, thereby precipitating plasmid DNA; and separating the precipitated plasmid DNA from the mixture, thereby obtaining isolated plasmid DNA. Another composition according to the invention contains an enzyme for digestion of cellular RNA, a stabilizer for protecting plasmid DNA from nuclease digestion, and a precipitating agent for precipitating plasmid DNA. Another method of isolating plasmid DNA from a bacterial culture comprises simultaneously digesting cellular RNA in a clarified lysate with an enzyme, protecting plasmid DNA from nuclease digestion, and precipitating plasmid DNA.