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:
- 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.
- 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.
- 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.
- 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.
- 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.