5 Minute Transformation Protocol for NEB® Stable Competent E. coli (C3040H/C3040I)

  1. C3040H: Remove cells from -80°C freezer and thaw in your hand. Place on ice.
    C3040l: 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.
  2. Add 1–5 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.
  3. Place the mixture on ice for 2 minutes. Do not mix.
  4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
  5. Place on ice for 2 minutes. Do not mix.
  6. 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.