5 Minute Transformation Protocol for NEB® Stable Competent E. coli (C3040)
- Remove cells from -80°C freezer and thaw in your hand.
- Thaw a tube of NEB Stable 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 1–2 µl containing 100 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 2 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
- Place on ice for 2 minutes. Do not mix.
- Pipette 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture. Immediately
spread 50–100 µl onto a selection plate and incubate overnight at
30°C for 24 hours.
Note: Plasmid selection using antibiotics other than ampicillin require an outgrowth period of 60 minutes at 30°C before plating on selective media. 30°C or 37°C may be used for plate incubation, however 30°C is recommended as some constructs may be unstable at elevated temperatures.