Preparation of Electrocompetent Bacteria
Please list modifications after the protocol.
- 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.
- 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.
- 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.
- 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.
- Repeat step 4 once.
- 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).
- Wash pellet with 50 ml cold, sterile H2O. Spin as in step 6. (Crush some dry ice in an ice bucket).
- Resuspend pellet in 40 ml of cold, sterile 10% glycerol. Spin as in step 6.
- 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).
- 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.