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.