Team:Tuebingen/Notebook/Protocols/competent cells

Creation of competent Escherichia coli cells

It is generally to be considered to keep the bacteria at 0 °C. The equipment has to be cooled on ice before usage.

1. 2.0 ml of a bacterial pre-culture are transferred to 100 ml of 37 °C warm LB medium
2. Incubate at 37 °C until the culture reaches a OD600/ml of ca. 0.5
3. Transfer the culture with the required OD in 50 ml culture tubes
4. Centrifuge for 5 min with 5000 rpm at 4 °C
5. Resuspend the pellet in 15 ml cold and sterile 100 mM CaCl2 and incubate for 30 min on ice
6. Centrifuge for 5 min with 5000 rpm at 4 °C
7. Resuspend the pellet in 4 ml cold and sterile 1 M CaCl2 and incubate for 15 min on ice
8. Add 840 μl 87 % (w/v) glycerine (end concentration of 15 %)
9. Aliquot 200 μl of the cell suspension in Eppendorf-tubes and freeze in liquid nitrogen. Store at -80 °C.