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 #E1203. Important – please read the FAQs regarding competent cell requirements before using a different cell strain.
- Thaw a 50 μl tube of competent cells on ice for 10 minutes.
- Add 2 μl of ligation reaction; gently mix by flicking the tube 4–5 times.
- Incubate on ice for 20 minutes.
- Heat shock at 42°C for 30 seconds.
- Chill on ice for 5 minutes.
- 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 www.neb.com/C3019
. The cloning efficiency cells provided with the NEB PCR Cloning Kit (NEB #E1202
) differ from the high-efficiency strain in their transformation efficiencies only.