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Preparation and Transformation of Competent E. Coli

Adapted from Inoue et al: High efficiency transformation of Escherichia coli with plasmids. Gene, 96(1990).

Preparation of the cells

  1. Grow 1 colony of cells in 25 ml of SOB medium for 6-8 hours at 37°C.
  2. With this starter culture, inoculate 250ml of SOB medium in the afternoon. Inoculate with 2 ml. Grow O/N at room temperature.
  3. The next day, continue growing the cells until the OD600 reaches 0.55.
  4. Transfer the cells to an ice-water bath for 10 minutes.
  5. Harvest the cells by centrifugation at 2500g for 10 minutes at 4°C.
  6. Pour of the supernatant, and resuspend in 80 ml of ice-cold Inoue transformation buffer (by swirling, if you have that much patience).
  7. Harvest the cells by centrifugation at 2500g for 10 minutes at 4°C.
  8. Resuspend the cells in 20 ml of ice-cold Inoue transformation buffer.
  9. Add 1.5 ml of DMSO, mix, and keep on ice for 10 minutes.
  10. Dispense aliquots (50 µl per transformation) into pre-chilled microfuge tubes, and snap-freeze in liquid nitrogen. Store at -80°C.

Transformation

  1. Thaw a tube of cells on ice, and transfer to 17 x 100-mm polypropylene tubes (on ice).
  2. Add DNA to the cells (max 5% of the total volume).
  3. Keep the tubes on ice for 30 minutes.
  4. Heat shock at 42°C for 90 seconds in a water bath, then cool on ice for 1-2 minutes.
  5. Add 500 µl of SOC medium, and incubate for 1 hour in a 37° shaker.
  6. Plate 250 µl on plates with appropriate antibiotics.

Inoue Transformation Buffer

  1. Prepare 0.5 M PIPES, adjust pH to 6.7 with KOH.
  2. Mix the following:
    Ingredient Amount Concentration
    MnCl2·4H2O 10.88 g 55 mM
    CaCl2·2H2O 2.20 g 15 mM
    KCl 18.65 g 250 mM
    PIPES (0.5 M, pH 6.7) 20 ml 10 mM
    H2O to 1 L
  3. Store at -20°CSterilize by filtration through a prerinsed 0.45µm filter.