Transformation Protocol (C3032)

Protocol

1. Thaw a tube of SHuffle Competent E. coli cells on ice until the last crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
2. 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.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 30°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 30°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50–100 µl of each dilution onto a selection plate and incubate overnight at 30°C. Alternatively, incubate at 25°C for 48 hours.