Team:Groningen/Protocols and Protocols/Heat shock transformation
Blue Bio Energy
Heat Shock Transformation
Standard heat shock transformation.
ML-I
Thaw
Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
Add plasmids
Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Mix carefully by gently swirling the pipettip in de mixture for 2 rounds.
Place on ice
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock
Heat shock at exactly 42°C for exactly 2 minutes. Do not mix.
Ice again
Place on ice for 2 minutes. Do not mix.
Add LB
Pipette 900 µl of room temperature LB media into the mixture.
Incubate and shake
Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
Preheat plates
Preheat the media plates (with corresponding antibiotics) to 37°C.
Spin
Spin down the cells at 8000 rpm for 1 min and remove the supernatant. Resuspend the pellet in 100 µL LB.
Plate
Spread the 100 µl on a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or RT over weekend.