Preparation of bacteria super competent cells

1. Prepare 2.5 ml of preculture of bacteria for 24 hours.

2. In an Erlenmeyer flask of two liters, inoculate 250 ml of LB with 1 ml of fresh preculture.

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

4. Let bacteria cells grow at 20 °C with agitation at 180 rpm by incubator for 24 h.

5. The final optical density of the solution is measured at tfin by spectrometer.

6. Incubate the solution at 0 °C for 10 min.

7. Centrifuge the solution for 10 min at 4 °C and 6000 rpm.

8. Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer, incubate for 10 min at 60 °C.

9. Centrifuge the solution for 10 min at 4 °C and 6000 rpm.

10. Remove and discard supernatant, resuspend the pellet in 15 ml ice-cold Transfo Buffer, add 1 ml DMSO for a final concentration of 7%.

11. Incubate the solution at 0 °C for 10 min.

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