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Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient
Authors:S J Garger  O M Griffith  L K Grill
Affiliation:1. Zoecon Corporation, 975 California Avenue, Palo Alto, California 94304 USA;2. Beckman Instruments, Inc., Spinco Division, 1050 Page Mill Road, Palo Alto, California 94304 USA
Abstract:A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.
Keywords:SDS  sodium dodecyl sulfate  TE buffer  10 mM Tris-HCl, pH 8.0, 1 mM EDTA  refractive index
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