Preparation of Electrocompetent E. coli

Preparation of Electrocompetent E. coli

• Inoculate 100 mL overnight culture grown at 30​​ C into 3 × 1L LB cultures in 3 Fernbach baffled flasks (OD600 ~ 0.05).

• Incubate at 37˚C, 200 rpm until OD600 reaches 0.3 – 0.4 (~1.5 – 2h).

• Chill 250 × 0.6 mL sterilized tubes1​​ by incubating at –80 ˚C in racks (wrap with Saran Wrap).

• Incubate on ice water bath with swirling for 30 min.

• Spin at 8,500 x g in chilled 12 x 250 mL bottles for 8 min. Decant supernatant. Resuspend in 10% glycerol and combine in pellets into 6 × 250 mL chilled bottles.

• Spin 10 min at 8,500 x g. Decant supernatant. Resuspend each bottle in 250 mL chilled 10% glycerol.

• Spin 10 min at 8,500 x g. Decant supernatant. Resuspend in 6 × 50 mL chilled 10% glycerol in a chilled 50 mL Falcon tube.

• Spin 10 min at 8,500 x g. Decant supernatant. Resuspend in 6 × 0.5 mL. Pool all of the cells into a single tube.

• Measure OD600 in triplicate using a 1:200 dilution

• Dilute (or concentrate) the cells to OD600 ~ 100.

• Measure OD600 of 1:200 dilution (OD600 ~ 0.5 for 1:200 dilution).​​ 

• Aliquot 40 uL into pre-chilled 0.6 mL tubes. Freeze by storing at –80​​ C.

• Test for contamination of Cm, Cb, Km, and Sp resistant bacteria.

• Test that cells are reasonably competent.

1​​ If possible use colored tubes (we prefer green)