The following protocol results in only 10% efficiency compared to the High Efficiency Transformation Protocol. For C2992H, perform steps 1-6 in the tube provided.
- C2992H: Remove cells from -80°C freezer and thaw in your hand.
C2992I: Thaw a tube of competent E. coli cells on ice for 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 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 SOC into the mixture. Immediately spread 50-100 μl onto a selection plate and incubate overnight at 37-42°C. NOTE: Selection using antibiotics other than ampicillin may require some outgrowth before plating on selective media. Colonies develop faster at temperatures above 37°C, however some constructs may be unstable at elevated temperatures.