https://books.google.com.bd/books?id=2QovDwAAQBAJ&pg=PA60&lpg=PA60&dq=Can+BL21(DE3)+grow+in+30+°C+following+electroporation?&source=bl&ots=ZQVh16pIG8&sig=ACfU3U0RfXHZEVoKo2otq0GKmyf4yu3GeQ&hl=en&sa=X&ved=2ahUKEwj6mLWpyI_pAhVGxjgGHYcIC_0Q6AEwBXoECAoQAQ#v=onepage&q=Can%20BL21(DE3)%20grow%20in%2030%20°C%20following%20electroporation%3F&f=false

Dear Davis

Theoretically E.coli can growth but slowly at 30°C than 37°C therefore is possible that an O/N incubation is not necessary to obtain visible colonies and you need to incubate longer time.

However i see one possible bugs in your protocol, contaminants such as salts and proteins can lower electroporation efficiency therefore for Electroporation the plasmid need to be in water.

You can remove the salts by dializing the plasmid drop before elettroporation by by sticking the DNA drop in a membrane as showed in the attached file.

https://www.capitolscientific.com/lab-supplies/Filtration-Products/Membrane-Filters/Plain-Membrane-Filters/Millipore-Plain-Membrane-Filters/Millipore-MF-Millipore-DNA-Fillter-Paper-for-Dialysis-of-DNA-and-Proteins

good luck

Manuele

• Each strain has its own electroporation stress resistance and it results in different electroporation efficiencies. In my experience BL-21 are not the best electroporable cells. The plasmid also contributes. Some plasmids are better for transformation than others. I don't see any gross mistake in your procedure.
• There are two things indicating that your electrocompetent cells are suffering too much in the process. Time constant is low (values approaching 5 are usually found in successful electroporations) and the cells are recovered as chunks, which is not a good sign. Make sure that the cells has been properly conserved at -80 w/o freezing/defrosting in the mentime and do the electroporation procedure as quickly and gently as possible. I would include a control with another (not temp sensitive plasmid) to make sure that the cells are useful. If the cells do not work with any plasmid, perhpas you would need to buy fresh electrocompetent cells or alternatively prepare them at the lab, which is not that difficult.
• In any case, ways to increase electroporation efficiency include using more cells (up to 50 ul in 1mm cuvettes and up to 200 ul in 2mm cuvettes, with the latter voltage can be increased up to 2.5 kV) and more DNA (DNA can be scaled up but is preferable to have it in pure water). You could also grow in the plates a bigger volume of the electroporated cell suspension (even plate everything instead of steaking a bit). All the above described tricks will increase the chances of getting colonies, even if the host cell/plasmid combination is not the best for a highly efficient electroporation.

Dear Gertrudis Rojas and Manuele Martinelli, Thank you for your helpful advice. I have carried out a new electroporation, trying to be as gentle as I can with the cells and also trying to incubate them with the plasmid for ~20 minutes before electroporation. I also spread 100 ul on a plate after the procedure instead of just streaking. Sadly, they didn't grow out in 2 days.

I don't think that salts would be the problem, the plasmid is in TE which contains EDTA, and is regarded as suitable for electroporation.

Trying a larger volume of cells might be a good idea, unfortunately Sigma provides them in 25 ul aliquots, and I only have 3 left. I think I'll grow them out and create larger electrocompetent stocks.

I'll aim for a better time constant next time (~ 5 ms). I have been doing electroporation with a protocol that requires capacitance, resistance, voltage and cuvette size, but there is also a protocol that only needs voltage and time constant, maybe that would be better.

Another thing that came to my mind is whether my plasmid and bacterium are even compatible with each other. I am using a plasmid that has a pSC101 ori and a temperature sensitive rep101 gene. I don't see why these shouldn't work in a BL21(DE3) cell without IPTG induction. Maybe I'm missing something?

If you prepare your own electrocompetent cells, test them with another plasmid, just in case the nature of your plasmid is creating a problem. Then you will be sure that the cells and the protocol are fine.

I normally used in the past 1,8 kV (for 1mm cuvettes), 200 ohm and 25 uF, but the conditions might depend on the electroporator. Check the instructions of the one you have. In my current lab I have simple electroporators for microorganisms (Biorad and Eppendorf), with which you only need to set voltage, and time constants are also near 5ms.

TE-plasmids can be used to electroporate, but if you need to increase DNA amount (up to 1 ug) to achieve a successful electroporation by brute force, it may be necessary to have the plasmid in water.

You could also try transformation by heat shock instead of electroporation. Its much less efficient, but if you have a problem with your electroporator, this could be a way to circumvent it. Are you sure that your electroporator is working? I had a similar problem in my lab and the reason was an electrical problem in the machine, even though it was apparently working.

There are many useful suggestions above, but be sure you are giving it sufficient incubation time on the plates. At 30 C you might need 24 hours to see good colonies instead of overnight.