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.