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
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.
- Resuspend pellet in 20 ml lowMho solution.
- 5' x 3,000 g in a refrigerated centrifuge.
- Discard the supernatant each time, including the last.
Salmonella strains are best used at once and should not be
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
- 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
10% glycerol in deionized distilled H2O
Sterilize in autoclave.
Last Update: Thursday, 19-Jun-2014 11:50:38 PDT
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