Protocols

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.