'You vs. the Guy She Tells You Not to Worry About': Heat-Shock vs. Electroporation

I compared the results from identical cloning protocols preceding either a heat-shock or electroporation bacterial transformation into NEB competent E. coli cells (chemically competent or electrocompetent) and found that the electroporation transformation consistently resulted in thousands more colonies than the heat-shock transformation. Getting millions of colonies are necessary for building large mutant libraries, but I think it also stands to reason that optimizing transformation efficiency increases the likelihood of obtaining one transformant if you are doing a complicated cloning protocol and are having trouble getting any colonies at all. Attached are pictures where 1/10 of the total outgrowth from each transformation was plated. The electroporation protocol was also much quicker than the heat-shock protocol. Here is what I did for the electroporation:

  1. I added 1.5μL of my newly-cloned plasmid (unpurified!) to 40μL of NEB 10-beta Electrocompetent E. coli in a new tube that I had previously chilled on ice, pipetted to mix gently, and let the mixture sit on ice for 1 minute.
  2. I transferred all of the mix to a 2mm cuvette that I had previously chilled on ice. I stuck the end of the pipette as far down into the cuvette as I could while dispensing the mix, and afterward, I tapped the cuvette on the bench a few times to get the cells to the very bottom of the cuvette.
  3. I electroporated the cuvette at ~2500V for ~5ms (1 pulse, exponential decay), and immediately added 1mL of SOC outgrowth media directly to the cuvette (no need to mix after and I made sure to add it super quickly). I made sure to check the electroporator afterward to confirm that the pulse had been the correct voltage and time I set it for.
  4. I left the cuvette to grow in the 37°C shaking incubator (~200 RPM) for 1 hour. You can also first transfer the cells to a culture tube first before leaving it in the incubator, but this isn't necessary.
  5. After the growth period was done I plated 100μL of it onto a pre-warmed plate of LB + AMP + NAT (as my plasmid has AMP and NAT selectable markers) and let it grow at 37°C overnight.

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Nice post! I've never tried to incubate the cells for outgrowth in the electroporation cuvette directly but always transfer them into a different tube - have you found that incubating the cuvette reduces its reusability? I currently reuse my cuvettes by soaking in Ethanol & UV-ing, and haven't had a problem yet but am worried that culturing bacteria in the cuvettes may change that.

Adrian Pascu3 months ago

Use CaCl2 like real men do! Electroporation is for p...s!

Nick Gervais3 months ago

Hey @David! I've never reused a cuvette, so I'm not sure. If you are applying ethanol and UV-ing I don't see why not, especially if your strain isn't forming biofilms and can be easily washed off.

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