2) Put 100 µL of competent bacteria in either an pre-cooled Eppendorf tube or a ... 5) Heat-shock the cells either at 37°C for 2 minutes or at 42°C for 45 seconds.
1) Thaw competent cells on ice (do not thaw at room temperature) 2) Put 100 µL of competent bacteria in either an pre-cooled Eppendorf tube or a pre-cooled 12 mL Falcon tube (recommended). 3) Add 2-5 µL of ligation/plasmid to the cells††. 4) Let stand on ice for 10 minutes‡‡. 5) Heat-shock the cells either at 37°C for 2 minutes or at 42°C for 45 seconds. 6) Put the bacteria back on ice for 2 minutes 7) Let the bacteria recover for 45 minutes - 1 hour§§ in 500 µL/1 mL of LB/SOC. Do not let the cells recover more than 1 hour to avoid bacterial division that will minimize the number of independent events. 8) Spread on agar plate containing the appropriate selection.
†† ‡‡
Ligation buffer inhibits transformation. The lowest the volume is the best it is. At this step, the cells can stand on ice more than 10 minutes.
