Transformation of competent E . coli cells with plasmid DNA
Thaw the appropriate amount of competent cells on ice .
Pre - chill the required number of empty 1.5 ml microcentrifuge tubes .
Pipet 100 µl aliquots of cells into the pre - chilled tubes .
Add 5 - 10 µl of a ligation reaction mix or 5 ng of pure plasmid DNA to each tube .
Mix gently !
Incubate the tubes of ice for 30 min .
Heat shock the cells for 45 sec at 42°C .
Place the tubes immediately on ice for at least 2 min .
Add 1000 µl of SOC medium to each tube and incubate for 1 hour at 37°C with shaking .
Transfer the cultures to 1.5 ml microcentrifuge tubes and spin for 1 min at 10000 x g .
Remove 800 µl of the supernatant and resuspend the pellet .
Plate out the suspension on a LB agar plate containing the appropriate antibiotic .
Incubate the plates overnight at 37°C .
