Preparation of supercompetent E.coli cells

1. Prepare the overnight culture of E.coli strain DH5alphaZ1 at 37C with shaking at 200-250rpm.

2. In an Erlenmeyer flask of two liters, inoculate 250ml of LB with 2.5ml of overnight culture.

3. The initial optical density of the solution is measured at t0 by spectrometer.

4. Grow bacteria at 20 °C with agitation at 200-250 rpm to an OD600nm of 0.4 to 0.5

5. Stop the solution on ice (0 °C) for 10 mins.

6. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).

7. Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer, incubate on ice for 10 mins.

8. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).

9. Remove and discard 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. Distribute the solution in 150 cold eppendorfs (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.