Preparation of quick competent cells

Protocol for preparation of quick competent cells of E. coli.

  • 10 ml LB in culture tubes (two).
  • 50 mM CaCl2 (prechilled)
  • 1.5 ml microfuge tubes.
All materials should be autoclaved.

  1. Culture wild type E. coli. in LB medium (5 to 10 ml) in a culture tube and grow overnight at 37°C.
  2. Take 1ml LB in microfuge tubes and add 10 ul of overnight grown culture to it.
  3. Incubate these culture ( 37°C, with shaking, 2.5 hours).
  4. Take out the tubes and keep in ice for 0.5 hr.  (from now, do not expose the cells to high temperatures.)
  5. Centrifuge the culture to pellet cells (7000 rmp, 3 min, 4°C)
  6. Discard the supernatant
  7. Resuspend pellet in 1 ml CaCl2 (50 mM)
  8. Centrifuge (7000 rmp, 3 min, 4°C)
  9. Repeat step 6,7,8 .
  10. Discard the supernatant.
  11. Resuspend pellet in 100 ul CaCl2 (50 mM).
These are your competent cells which are to be used immediately for transformation. I have not tried to use them after storing at -80°C but as there is no glycerol in it, most probably they won't survive.

P.S.: This protocol was told to me by one of my labmates and I am using it since then. 


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