Electroporation-Competent Cells

3/6/01 ECK
revised 8/6/2012


Description

Electrocompetent cells are normal cells suspended in a low conductivity medium which prevents arcing during electroporation. Always keep cells on ice, and drain pellets well after centrifugation! It is important to grow the cells to mid-log or earlier, especially for Salmonella.

Protocol

Growth
  • Inoculate 200 ml L-Broth (E.coli or Salmonella galE+ strain) or NB or Minimal/Glucose (Salmonella galE strain) with 2 ml of an overnight culture.
  • Incubate 3 hr with shaking at 37°.

  • Harvesting and Washing
  • Pellet cells 6' x 6,000 rpm in GSA rotor.
  • Repeat three times:
  • Vortex final pellet without added liquid until it forms a thick mud. The cells are now ready for use and should be kept on ice.

    Storage
  • Salmonella strains are best used at once and should not be frozen.
  • E.coli strains can be treated in the following fashion:

    Materials

    lowMho solution
    10% glycerol in deionized distilled H2O Sterilize in autoclave.
    Last Update: Thursday, 19-Jun-2014 11:50:38 PDT
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    Eric Kofoid eckofoid at ucdavis.edu