CompetentCell

Inoculate 100µL of E. coli in10mL of antibiotic-free starter LB culture medium. Incubate during at 37°C and 225rpm ,0h 0m 0s

Heat the LB media in the water bath at37°C

Inoculate 40mL of LB media with 1mL of cell culture from step 1.

Incubate at 37°Cwith constant agitation (225rpm during 0h 30m 0s).

Measure de optical density at 600 nm every 0h 30m 0s until an OD600 of 0.4-0.6 is reached.

Chill for0h 20m 0s On ice

Centrifuge during 0h 15m 0s at 4°Cand 3000rpm,0h 0m 0s

Discard the supernatant, add 20mL of solution 50 mM and resuspend carefully.

Chill for 0h 20m 0s On ice

Centrifuge during 0h 15m 0s at 4°Cand 3000rpm,0h 0m 0s

Discard the supernatant, add 5 mL of CaCl2 solution 100 mM and resuspend carefully. ChillOn ice for 0h 20m 0s

Preserve at 4°C