High Efficiency Transformation Protocol ( modified )
Warm SOC media to room temperature .
Thaw a tube of NEB 5 - alpha Competent E . coli cells on ice for 10 minutes .
Aliquot 25uL of the cells into a new tube .
Add 0.5 - 2.5 µl containing 0.5 pg - 50 ng of plasmid DNA to each cell mixture .
Carefully flick the tube 4 - 5 times to mix cells and DNA .
Do not vortex .
Place the mixture on ice for 30 minutes .
Do not mix .
Heat shock at exactly 42°C for exactly 30 seconds .
Do not mix .
Place on ice for 5 minutes .
Do not mix .
Pipette 475 µl of room temperature SOC into the mixture in each tube .
Place at 37°C for 60 minutes , shaking vigorously ( 250 rpm ) or rotating .
Warm selection plates to 37°C .
Mix the cells thoroughly by flicking the tube and inverting .
Perform several 10 - fold serial dilutions in SOC .
Spread 50 - 100 µl of each dilution onto a selection plate .
Incubate overnight at 37°C
