Team:BCCS-Bristol/Protocols-Preparation of electro competent cells

Preparation of electro competent cells

1. 5 ml LB were inoculated with a colony from a freshly streaked E. coli DH5α and incubated overnight at 37°C and 225 rpm
2. 4x 1 l flasks, each containing 100 ml LB, were inoculated with 1 ml overnight culture per flask, then incubated as before until the OD₆₀₀ reached 0.3 to 0.4 (ca. 3-4 h)
3. Cell tubes were decanted into 8x 50 ml falcon tubes and centrifuged at 2500 g in a Beckmann CS-6R bench top centrifuge for 10 min at 4°C. All manipulations henceforth were carried out with the cells on ice.
4. After discarding the supernatants, each cell pellet was resuspended in 50 ml ice cold sdw (sterile distilled water) and centrifuged as before.
5. After discarding the supernatants, the cells were resuspended in the remaining liquid and pooled into 4 tubes, then resuspended in 50 ml ice cold sdw and centrifuged as before.
6. After discarding the supernatants, the cells were resuspended in the remaining liquid and pooled into one tube, then resuspended in 50 ml ice cold sdw and centrifuged as before.
7. The single cell pellet was resuspended in 1.2 ml ice cold 20 % glycerol and divided into 50 µl aliquots, which were rapid frozen in liquid nitrogen and stored at -80°C.