The following protocol results in only 10% efficiency compared to the Transformation Protocol. For C2925H, perform steps 1-6 in the tube provided.
- For C2925H: Remove cells from -80°C freezer and thaw in your hand.
For C2925I: Thaw a tube of dam-/dcm- 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 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.