High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (C3019I)
Thaw a tube of NEB 10-beta Competent E. coli cells on ice until the last ice crystals disappear.
Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the 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 950 µl of room temperature SOC into the mixture.
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
