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).