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Making Electrocompetent Cells

Method

  1. Grow 5 ml O/N in LB.
  2. Inoculate 400-500 ml LB with 1/100 volume bacteria from O/N culture.
  3. Grow to OD600 = 0.6.
  4. Chill cells on ice for 15 minutes to several hours.
  5. Pellet cells at 4°C, 2600 rpm, GSA rotor.
  6. Wash 2X in 200 ml pre-chilled, unused, sterile H2O.
  7. Pellet as above.
  8. Resuspend in final volume of 2 ml GYT*.
  9. Freeze 50-µl aliquots immediately in dry ice/ethanol.
  10. Store -80°C.

*GYT

  • 10% Glycerol
  • 0.125% Yeast Extract
  • 0.25% Tryptone
  • Q with H2O
  • Autoclave and store at 4°C

Making Heat Shock Competent Cells

  1. Inoculate 500 ml LB with 5 ml of a 25-ml O/N culture.
  2. Grow at 37°C until OD600 = 0.45-0.55
  3. Chill cells on ice water for 2 hours.
  4. Pellet cells at 4°C, 2500 g (3700 rpm in GSA rotor) for 15-20 minutes.
  5. Resuspend cells in 10 to 20 ml ice-cold Ca/Mg buffer**.
  6. Then bring volume up to 500 ml with the Ca/Mg buffer.
  7. Incubate on ice for 45 minutes.
  8. Centrifuge cells 1800 g (3200 rpm GSA rotor) for 10 minutes at 4°C.
  9. Gently resuspend pellet in 50 ml ice-cold Mg/Cl buffer.
  10. Pool cells if you have split them up and add 80% glycerol drop by drop, on ice with gentle swirling, to a final concentration of 15% v/v.
  11. Aliquot cells (0.2-1 ml), freeze on dry ice and store at -80°C.

**Ca/Mg Buffer

  • 100mM CaCl2
  • 70mM MgCl2
  • 40mM Sodium Acetate pH 5.5
  • Filter sterilize

© 2014 The University of Texas MD Anderson Cancer Center