Electrocompetent Cells

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:

Resuspend pellet in 20 ml lowMho solution.
5' x 3,000 g in a refrigerated centrifuge.
Discard the supernatant each time, including the last.

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:

Sit a metal block for Eppindorf vials on dry ice. Load it with 600 µL Eppindorf vials.
Use electric pipetter to dispense 40 µL aliquots to the cold vials.
Close vials and store at -70°. In this form, cells are good for several months.

Materials

lowMho solution
10% glycerol in deionized distilled H2O Sterilize in autoclave.

Last Update: Thursday, 12-Dec-2024 00:55:20 UTC

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Eric Kofoid eckofoid at ucdavis.edu