Transformation Protocol
Thaw competent cells on ice .
Chill approximately 5 ng ( 2 μl ) of the ligation mixture in a 1.5 ml microcentrifuge tube .
Add 50 µl of competent cells to the DNA .
Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA .
Do not vortex .
Place the mixture on ice for 30 minutes .
Do not mix .
Heat shock at 42°C for 30 seconds .
Do not mix .
Add 950 µl of room temperature media to the tube .
Place tube at 37°C for 60 minutes .
Shake vigorously ( 250 rpm ) or rotate .
Warm selection plates to 37°C .
Spread 50–100 µl of the cells and ligation mixture onto the plates .
Incubate overnight at 37°C
