Transformation Protocol for NEB PCR Cloning Kit

The following protocol is designed for NEB 10-beta Competent E. coli (NEB #C3019) which are included in the NEB PCR Cloning Kit (NEB #E1202) only. Competent cells are also available separately for use with NEB #E1203Important – please read the FAQs regarding competent cell requirements before using a different cell strain.
  1. Thaw a 50 μl tube of competent cells on ice for 10 minutes.
  2. Add 2 μl of ligation reaction; gently mix by flicking the tube 4–5 times.
  3. Incubate on ice for 20 minutes.
  4. Heat shock at 42°C for 30 seconds.
  5. Chill on ice for 5 minutes.
  6. Add 950 μl of NEB™ 10-beta/Stable Outgrowth Medium and place at 37°C for 60 minutes with rotation or shaking at 250 rpm (outgrowth step).
Note: For further general discussion about transformations with NEB 10-beta Competent E. coli, please refer to the transformation protocol at The cloning efficiency cells provided with the NEB PCR Cloning Kit (NEB #E1202) differ from the high-efficiency strain in their transformation efficiencies only.