Template:Team:Groningen/CONTENT/Protocols and Protocols/Heat shock transformation

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.