Preparation of pure plasmid or cosmid DNA using single-strand affinity matrix and gel-filtration spin columns.
A rapid method has been developed for ultrapure plasmid or cosmid DNA isolation from ten-mL to several hundred-mL cultures of Escherichia coli (midi to maxi prep). A cleared lysate is prepared by alkaline lysis, followed by a quick alcohol precipitation step. Denatured bacterial DNA and RNA having at least 20 nucleotides of single-stranded regions are removed from the supercoiled plasmid by binding strongly to the single-strand affinity matrix (SSAMTM). Plasmid DNA is then effectively purified on a gel-filtration spin column to remove SSAM, proteins, small RNA and salts. This method produces consistent yields of high-quality plasmids that are suitable for use in many molecular biology applications. In addition, recombinant cosmids of approximately 46 kb can be purified intact, free of chromosomal DNA.