NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol

For use with NEBuilder® Hifi DNA Assembly Cloning Kit (NEB #E5520)and NEBuilder HiFi DNA Assembly Bundle for Large Fragments (NEB #E2623 )
  1. Thaw competent cells on ice.
  2. Add 2 μl of the chilled assembly product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4–5 times. Do not vortex.
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock at 42°C for 30 seconds. Do not mix.
  5. Transfer tubes to ice for 2 minutes.
  6. Add 950 ul of room temperature SOC medium to the tube. When using NEB 10-beta or NEB Stable E. coli competent cells, add 950 ul of the NEB 10-beta/Stable Outgrowth Medium.
  7. Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plates to 37°C.
  9. Spread 100 μl of the cells onto the selection plates. Use Amp plates for the NEBuilder Positive Control sample. Both the transformation control pUC19 and the NEBuilder Positive Control are plated on Amp plates.
  10. Incubate overnight at 37°C.