Team:Paris Saclay/Protocols/Preparation of supercompetent Ecoli cells

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Preparation of supercompetent E. coli cells

  1. Prepare an overnight culture of E. coli strain and incubate it at 37°C with shaking at 200-250 rpm.
  2. In a 2 liter Erlenmeyer flask, inoculate 250 ml of LB with 2.5 ml of overnight culture.
  3. The initial optical density at 600 nm of the solution is measured at t0 with a spectrometer.
  4. Grow bacteria at 20 °C with agitation at 200-250 rpm to an OD600nm of 0.4 to 0.5
  5. Place the culture on ice (0 °C) for 10 mins.
  6. Centrifuge the culture for 10 min at 4 °C at 3000 rpm (2500g).
  7. Remove and discard the supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer (see below), incubate on ice for 10 mins.
  8. Centrifuge the culture for 10 min at 4 °C at 3000 rpm (2500g).
  9. Remove and discard the supernatant, resuspend the pellet in 20 ml ice-cold Transfo Buffer, add 1 ml DMSO for a final concentration of 7%.
  10. Incubate on ice for 10 min.
  11. Aliquot the solution in chilled 1.5 ml microcentrifuge tubes (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.


Component of Transfo Buffer:
                        HEPES 10 mmol/L
                        MnCl2 55 mmol/L
                        CaCl2 15 mmol/L
                        KCl 250 mmol/L
                        KOH


Preparation of 500 ml of Transfo Buffer:
  • Dissolve 1.19 g HEPES, 1.10 g CaCl2, and 9.32g KCl in 500 ml of water,
  • Adjust the pH at 6.7 with KOH,
  • Add 5.44 g MnCl2,
  • Sterilize the solution by filtration and store the solution at 4°C.