Electrocompetent NEB turbo E. coli prep

I used this method to make electrocompetent NEB turbo E. coli cells. This protocol is based on the one from Barrick labs which is linked below.

https://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells

  • One colony of NEB turbo E. coli was taken from a petri dish and inoculated into 5 mL of LB broth, in a 15 mL conical tube, and placed in a 37 °C tube-turner incubator overnight.
  • Next, 100 mL of fresh LB broth was inoculated to an initial concentration of OD 0.071 using the overnight culture, and placed into a 37 °C incubator with magnetic stirring for ~2 hours.
  • After about two hours of incubation, the 100 mL liquid culture reached a final concentration of OD 0.503 which was measured using a turbidity spec.
  • The culture was then poured into two 50 mL conical centrifuge tubes and chilled using ice cold metal beads for ~5 minutes.
  • The tubes were centrifuged at 4,000 RPM for 5 minutes and the supernatant was discarded.
  • The pellets of cells were then each resuspended in 40 mL of 4°C 10% glycerol solution using a vortex mixer.
  • The tubes were then centrifuged again at 4,000 RPM for 5 minutes and the glycerol supernatant was discarded.
  • The cold glycerol washing-centrifuging process was repeated a total of four times.
  • Finally, the electro-competent cell pellets were each resuspended in 500 μL of 10% glycerol solution, divided into 50 μL aliquots, and placed into a -80 C freezer for storage.
  • (I’m working from a university lab at the moment. Under normal circumstances I work from my home lab and do not have access to such a cryo-freezer, so I would usually just use the electrocompetent cells right away without delay.)

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Nick Gervais4 months ago

I was actually looking for a new protocol for electrocompetent cells since the transformation efficiency with mine is terrible. What concentration/volume do you prepare your vector to before transforming into these cells?

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