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From 2010.igem.org

Making competent cells

Materials:

- bacterial culture

- centrifuge

- 0.1 M MgCl2

- 0.1 M CaCl2

- 80% glycerol

Protocol:

1. Cultivate 100 mL LB medium, 37 °C o/n with shaking(140 rpm)

2. Cultivate 50 mL further in 800 mL LB, ~ 2 hours at 37 °C with shaking (140 rpm) until OD601 = 0.4

3. Centrifuge 4500 rpm, 6 min, 4 °C

4. Resuspend pellet in 80 mL ice-cold 0.1 M MgCl2

5. Centrifuge 4500 rpm, 6 min, 4 °C

6. Resuspend pellet in 50 mL ice-cold 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet, solution should look like milk)

7. Incubate 15 min on ice

8. Centrifuge 4500 rpm, 6 min, 4 °C

9. Resuspend pellet in 15 mL 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet)

10. Divide in several eppendorf tubes (~ 10)

11. Incubate 60 min on ice

12. Centrifuge 14000 rpm 10 min 4 °C

13. Add ~200 µL ice-cold 80% glycerol per eppendorf tube (depends on the amount of pellet)

14. Put all the solutions together and divide in parties of 100 µL and quickly put them in the liquid nitrogen

15. Freeze in -80 °C