Maheshri:CompetentE

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Preparation of Electrocompetent Bacteria

Please list modifications after the protocol.

  1. Inoculate 0.5-1.0 L of LB (no antibiotics) with 5 ml of a fresh overnight culture (for example DH5alpha). Put sterile H2O (0.5-1.0 L) at 4°C to cool.
  2. Grow cells at 37°C with vigorous shaking to an OD600 of (approx.) 0.6. (This usually takes around 3-4h). Put 1.5 ml eppendorf tubes (50-60 for 1L) at 4°C to cool. Put 10% sterile glycerol (50 ml at least) at 4°C to cool.
  3. To harvest cells, chill flask in ice for 15-30 minutes. At this time also chill 2-4 sterile 250 ml centrifuge bottles. After incubating on ice, spin cells in Sorvall RC 2-B at 5-6,000 rpm (4000 x G max) for 15 minutes.
  4. Combine pellets into 1 or 2 tubes (if 2 or 4 tubes used in previous step, respectively). Resuspend with 250 ml cold, sterile H2O per 500 ml of starting culture . Repeat spin as in step 3.
  5. Repeat step 4 once.
  6. Resuspend each pellet in 10 ml of cold, sterile H2O. Combine pellets into one pre-chilled 50 ml Falcon tube. Add cold, sterile H2O to a final volume of 50 ml. Spin cells in a pre-chilled Beckman tabletop centrifuge at 3,000-3,500 rpm. (Alternatively, wait until next wash to combine pellets).
  7. Wash pellet with 50 ml cold, sterile H2O. Spin as in step 6. (Crush some dry ice in an ice bucket).
  8. Resuspend pellet in 40 ml of cold, sterile 10% glycerol. Spin as in step 6.
  9. Very carefully pour off glycerol. Estimate the volume of the cell pellet. Add an equal volume of cold, sterile 10% glycerol. Mix with a chilled blue tip (for P-1000).
  10. Immediately aliquot cells into pre-chilled, labeled 1.5 ml eppendorf tubes (on wet ice). Use a pre-chilled Combitip (sterile) to aliquot cells. As quickly as possible, place cells onto crushed dry ice. Aliquot cells in 50 µl volumes into tubes.
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