Preparation of ultra-competent E. coli cells for transformation

Based on Inoue et al (1990), Gene, 96:23-28, with modifications.

  1. Culture cells (DH5a in my case) on LB agar plate at 37oC overnight.
  2. Pick up 10 -12 large colonies and culture in 250ml SOB in a 1L flask, 19oC with vigorous shaking to OD600=0.5 (normally it takes 24-36hrs)
  3. Place the flask in ice for 10 min.
  4. Pelleting the cell by spining at 4000rpm for 10 min at 4oC.
  5. Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.
  6. Spin at 4000rpm for 10 min at 4oC
  7. Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO (the DMSO needs to be stored at -20oC o/n before use).
  8. Aliquote the cell to 50 to 500ul for transformation or store at  -70oC

    *         Note: The E. coli cells prepared this way are normally 100 to 1000 times more efficient than normal calcium method, so do not plate too dense!

  • SOB solution:

    *         0.5% yeast extract
    *         2% tryptone
    *         10mM NaCl
    *         2.5mM KCl
    *         10mM MgCl2
    *         10mM MgSO4
    *         Dissolve in nanopure water and autoclave to sterilize.

  • TB solution:
    *         10mM PIPES
    *         15mM CaCl2
    *         250mM KCl
    *         Dissolve in nanopure water and adjust pH to 6.7 with KOH or
    HCl and then add MnCl2 to 55mM, and adjust to final volume. Sterilize by
    filtration with 0.45um filter and store at 4oC