Abstract
A method is described for the preparative purification of supercoiled DNA of bacterial cell extracts without an ultracentrifugal step. DNA of crude cell lysates, containing supercoiled plasmid DNA and chromosomal DNA fragments is freed of protein by chromatography on Sepharose B4. The separation of closed-circular supercoiled DNA from linear and open-circular DNA is performed by chromatography on hydroxyapatite in the presence of ethidium bromide by taking advantage of the different binding ability of the dye for linear and closed-circular DNA. The best separations are obtained when the linear or nicked DNA in the sample is complexed at a frequency of one dye molecule per three-five base-pairs. The extent of separation seems to depend only on the intrinsic superhelical density of the supercoiled DNA molecules and not on their sizes.
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