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== 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 80 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 40 mL 0.1 M CaCl2 (do not vortex, stir with Pasteur pipet) 10. Incubate 60 min on ice 11. Centrifuge 4500 rpm 10 min 4 °C 12. Add ~1 à 2 mL (depends on the amount of pellet) ice-cold 80% glycerol 13. Divide in parties of 30 µL and quickly put them in the liquid nitrogen 14. Freeze in -80 °C

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