Preparing Bacterial Genomic DNA.

Abstract

We describe two alternative procedures for purifying bacterial chromosomal DNA. The first procedure incorporates the use of a commercial kit based on silica membrane technology. This approach relies on the selective binding of DNA to a silica-based column in the presence of chaotropic salts (guanidine salts). Polysaccharides and proteins do not bind well to the column and flow through. Their residual traces, along with the guanidine salts, are removed during alcohol-based wash steps. The DNA is then selectively eluted under low-salt conditions. This method is quick and easy and yields genomic DNA suitable for most downstream applications. The second procedure, implemented for many years in our laboratory before the appearance of commercial kits, is based on the ability of the cationic detergent cetyl trimethyl ammonium bromide (CTAB) to complex with polysaccharides and proteins, producing an emulsion that can be removed by chloroform-isoamyl alcohol extraction. This procedure is therefore especially suited to working with Gram-negative bacteria, which typically produce large amounts of polysaccharides. Its main advantage, besides cost-effectiveness, is the high yield of the DNA obtained; its main disadvantage is that the workflow is relatively cumbersome.