High Efficiency Transformation for NEB® Stable Competent E. coli (C3040I)

  1. 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 -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.
  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. Place on ice for 5 minutes. Do not mix.
  5. Pipette 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture and place at 30°C for 60 minutes. Shake the tube horizontally at 250 rpm or rotate.
  6. Warm selection plates to 30°C.
  7. Mix the cells thoroughly by flicking the tube and inverting. Then spread 50-100 µl of cells or diluted cells onto a selection plate. Incubate plates 24 hrs at 30°C or overnight at 37°C.
Note: For best clone stability, incubate plates and liquid cultures at 30°C and prepare plasmid DNA from fresh transformants (from plates not greater than 3 days old). Store unstable clones as plasmid DNA, rather than cell-based glycerol stocks.