Control of pH during plasmid preparation by alkaline lysis of Escherichia coli.

Alkaline lysis of Escherichia coli is usually the method of choice for plasmid preparation, but ''ghost bands" of denatured supercoiled DNA can result if the pH is too high or the period of lysis is too long. By replacing the usual sodium hydroxide lysis solution with an arginine buffer prepared in the range of pH 11.4 to 12.

A nonalkaline method for isolating sequencing-ready plasmids.

We describe a simple method of isolating plasmid DNA directly from Escherichia coli culture medium by addition of lithium acetate and Sodium dodecyl sulphate, followed by centrifugation and alcohol precipitation. The plasmid is sufficiently pure that it can be used in many enzyme-based reactions, including DNA sequencing and restriction analysis. Chromosomal DNA contamination is significantly reduced by pretreatment of the culture with DNase I, suggesting that much of the contaminant is associated with permeable dead cells.