• My NEB
  • Print
  • PDF
  • 5 Minute Transformation Protocol (C3040)

    1. Remove cells from -80°C freezer and thaw in your hand. 
    2. 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.
    3. 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.
    4. Place the mixture on ice for 2 minutes. Do not mix.
    5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
    6. Place on ice for 2 minutes. Do not mix.
    7. Pipette 950 µl of room temperature SOC into the mixture. Immediately spread 50–100 µl onto a selection plate and incubate overnight at 30°C for 24 hours.

      Note: Selection using antibiotics other than ampicillin may require some outgrowth at 30°C before plating on selective media. Colonies develop faster at 37°C, however some constructs may be unstable at elevated temperatures.