Unit

UNIT 2.4 Preparation of Genomic DNA from Bacteria

  1. Kate Wilson

Published Online: 1 NOV 2001

DOI: 10.1002/0471142727.mb0204s56

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Wilson, K. 2001. Preparation of Genomic DNA from Bacteria. Current Protocols in Molecular Biology. 00:2.4.1–2.4.5.

Author Information

  1. Australian Institute of Marine Science, Townsville, Australia

Publication History

  1. Published Online: 1 NOV 2001
  2. Published Print: OCT 2001

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.