The Enrichment of Plasmid Dnas,in Bacterial Cell Lysates,Using an Alkaline-pH Procedure that Does Not Permanently Denature Them.

Abstract

The method described permits the enrichment of large (greater than or eoual to 54 kb), small (less than or equal to 2.1 kb), or intermediate sizes of plasmid DNAs. It is a modification of the plasmid enrichment method described by Currier and Nester (Anal. Biochem., 76, 431-441, 1976) in that it defers the alkali denaturation step until the pH and temperature can be controlled. This prevents permanent alkali denaturation of some plasmids. In general, total cellular nucleic acids are precipitated with either ethanol or isopropanol after lysates, in 3% w/v NaCl, are extracted with a phenol-chloroform mixture. The nucleic acids are then treated at an alkaline-pH (12.3-12.4), in a buffer, at 0°C, for a minimum time of 15 min. Denatured DNA, in 3% w/v NaCl, is removed with phenol. The RNA- containing, plasmid enriched fraction, is once again precipitated with either ethanol or isopropanol, dried in a vacuum, and redissolved. Samples are then digested with various restriction enzymes and/or examined directly on agarose gels after treatment with RNAase A.