Preparation of Genomic <scp>DNA</scp> from Bacteria

TLDR

Typical bacterial genomic DNA protocols involve lysis, protease incubation, and multiple extractions, but they fail to remove exopolysaccharides that inhibit restriction enzymes and ligases. The method adds a CTAB extraction after protease treatment to complex and remove polysaccharides and residual protein, and can be scaled with CsCl gradient purification for high‑purity DNA. This approach yields digestible chromosomal DNA from diverse gram‑negative bacteria that normally produce large amounts of polysaccharides.

Abstract

Abstract Most protocols for the preparation of bacterial genomic DNA consist of lysis, followed by incubation with a nonspecific protease and a series of extractions prior to precipitation of the nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in removing exopolysaccharides which can interfere with the activity of enzymes such as restriction endonucleases and ligases. In this unit, however, the protease incubation is followed by a CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein, effectively removing both in the subsequent emulsification and extraction. This procedure is effective in producing digestible chromosomal DNA from a variety of gram‐negative bacteria, all of which normally produce large amounts of polysaccharides. If large amounts of exceptionally clean DNA are required, the procedure can be scaled up and the DNA purified on a CsCl gradient, as described in the alternate protocol.