Electrocompetent cell preparation and transformation
Electrocompetent cells are prepared with the following protocol: (1) inoculate a single colony in a 10 mL tube for overnight growth; (2) transfer 1% V/V strains into a 500 mL shake flask with 100 mL LB liquid medium; (3) when optical density at 600 nm (OD600) of the bacterial culture reaches 0.4-0.6, place it on ice and chill for 30 min; (4) spin in centrifuge at 4000 rpm for 10 min, discard the supernatant, and add 40 ml ddH20 to wash the cell pellet; (5) spin in centrifuge at 4000 rpm for 10 min, discard the supernatant, add 40 ml 10% V/V glycerol to wash the pellet, and place it on ice for 10 min; (6) repeat step (5) with 20 ml then 10 ml 10% V/V glycerol, successively; (7) spin in centrifuge at 6000 rpm for 10 min, discard the supernatant, and add 2 ml 10% V/V glycerol to resuspend the pellet; (8) distribute the final dilutions (100 μl each) into the sterile 1.5 ml tubes, freeze it immediately with liquid nitrogen and store at − 80 °C.
About 250ng of plasmid was pipetted into 100 μl thawed electrocompetent cells, and transferred to a pre-cooled 1 mm cuvette (Bio-Rad micro pulser, 2.5 kV, 100 Ω, 25 μF). After pulse, we added 600 μL pre-warmed LB liquid medium (containing 30 μg/ml anhydrotetracycline or 10mM L-arabinose if necessary) and recovered it in tube at 37 °C for 1.5 hours. Finally, we spread this culture onto LB agar plate containing 100 μg/ml ampicillin and incubated it at 37 °C overnight. The transformation efficiency was calculated as the number of colony forming units per 1 μg of plasmid (cfu/μg).