Miniprep double-stranded DNA isolation

The standard method for the miniprep isolation of plasmid DNA includes the same general strategy as the large scale isolation. However, smaller aliquots of antibiotic containing liquid media inoculated with plasmid-containing cell colonies are incubated in a 37degC shaker for 12-16 hours. After collecting the plasmid containing cells by centrifugation, the cell pellet is resuspended in a hypotonic sucrose buffer. The cells are successively incubated with an RNase-lysis buffer, alkaline detergent, and sodium acetate. The lysate is cleared of precipitated proteins and membranes by centrifugation, and the plasmid DNA is recovered from the supernatant by isopropanol precipitation. The DNA is crudely checked for concentration and purity using agarose gel electrophoresis against known standards. A typical yield for this method of DNA isolation is 10-15 ug of plasmid DNA from a 6 ml starting culture.

Since highly supercoiled DNA is desired for double-stranded DNA sequencing, a modification of this method employing diatomaceous earth (19-22) sometimes is used for isolation of double-stranded templates for DNA sequencing with fluorescent primers. After removal of the precipitated proteins and membranes, the plasmid containing supernatant is incubated with diatomaceous earth and guanidine hydrochloride and this mixture is added into one of the twenty-four wells in the BioRad Gene Prep Manifold. The supernatant is removed by vacuum filtration over a nitrocellulose filter. The DNA-

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