Rapid protocol for preparation of electrocompetent Escherichia coli and Vibrio cholerae

J Vis Exp. 2013 Oct 8:(80):50684. doi: 10.3791/50684.

Abstract

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Bacteriological Techniques / methods*
  • Electroporation / methods*
  • Escherichia coli / cytology
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • Escherichia coli / physiology*
  • Transformation, Bacterial
  • Vibrio cholerae / cytology
  • Vibrio cholerae / genetics
  • Vibrio cholerae / growth & development
  • Vibrio cholerae / physiology*