Preparation of Genomic DNA from Bacteria

Kate Wilson1

1 Australian Institute of Marine Science, Townsville, Australia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.4
DOI:  10.1002/0471142727.mb0204s56
Online Posting Date:  November, 2001
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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‚Äźnegative 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.

     
 
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Table of Contents

  • Basic Protocol 1: Miniprep of Bacterial Genomic DNA
  • Support Protocol 1: Removal of Polysaccharides from Existing Genomic DNA Preps
  • Alternate Protocol 1: Large‐Scale CsCl Prep of Bacterial Genomic DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Miniprep of Bacterial Genomic DNA

  Materials
  • TE buffer ( appendix 22)
  • 10% sodium dodecyl sulfate (SDS)
  • 20 mg/ml proteinase K (stored in small single‐use aliquots at −20°C)
  • 5 M NaCl
  • CTAB/NaCl solution
  • 24:1 chloroform/isoamyl alcohol
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • Isopropanol
  • 70% ethanol

Support Protocol 1: Removal of Polysaccharides from Existing Genomic DNA Preps

  Additional Materials
  • Cesium chloride
  • 10 mg/ml ethidium bromide
  • CsCl‐saturated isopropanol or H 2O‐saturated butanol
  • 3 M sodium acetate, pH 5.2
  • Beckman JA‐20 rotor or equivalent
  • 50‐ml Oak Ridge centrifuge tubes
  • Wide‐bored pipet
  • 4‐ml sealable centrifuge tubes
  • Beckman VTi80 rotor
  • 3‐ml plastic syringe with 15‐G needle
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Figures

Videos

Literature Cited

Literature Cited
   Meade, H.M., Long, S.R., Ruvkun, C.B., Brown, S.E., and Ausubel, F.M. 1982. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J. Bacteriol. 149:114‐122.
   Murray, M.G. and Thompson, W.F. 1980. Rapid isolation of high‐molecular‐weight plant DNA. Nucl. Acids Res. 8:4321‐4325.
   Silhavy, T.J., Berman, M.L., and Enquist, L.W. 1984. Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
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