I tried the recipes below in two cells lines:
1.DPBS
2.DPBS+MgCl2(1mM)
3.DPBS+Sucrose(250mM)
4.DPBS+MgCl2(1mM)+Sucrose(250mM)
5.R Buffer (Neon)
the results showed that R buffer is the best one in both suspension and adherent cells. but the efficiencies of other buffers were not good in adherent cells. so i wonder if anyone have better recipes...
I have tried all the buffers you have tried. Personally I found that #4 worked just as well as the R buffer for our specific adherent lines (mostly HEK293T cells and some brain endothelial cells), but I noticed the cells were not behaving in the neon tip like they would with R (can't remember exactly what it was). The other 3 did not work as well for efficiency. Viability wise, anything with sucrose worked better than the R buffer. Sucrose makes it harder to clean the tubes and tips for reuse though..
So I thought it over and realized they probably used a potassium heavy buffer to prevent influx/efflux during electroporation, especially with large plasmids. Inside of cell should be high in [K+] and low in [Na+], so if you poke holes in the membrane and the outside media is PBS, you may get significant influx/efflux which can be bad. In the case of large plasmids (+5k), this could be very bad since the plasmid stays stuck in the membrane for a few minutes leaving large pores for flux. This is bad for viability and possibly efficiency since only non-transfected cells will plate/grow. My theory based on some experiments I did anyways.. While I did not find that this buffer significantly improve efficiency/viability, I should note that by the time I started playing around with the potassium concentration I had already developed electro conditions for good viability and +90% efficiency. Stuck with this recipe anyways since I thought it was theoretically better.
The buffer recipe I attached is worth a try if you have extra cells and time, because other than that I'm not sure what else to try to improve your efficiency.
There's a paper that suggests higher efficiency if you have a post electroporation RT incubation time of +5 minutes. Have you tried this? (Overcoming the Specific Toxicity of Large Plasmids Electrotransfer in Primary Cells In Vitro). I have seen viability shoot up (+80% in one case) after 5-10 min, but efficiency was already high so could not see a difference.
Best of luck, let me know if the SK buffer recipe matched R buffer performance. And try the SK buffer as your E buffer, too, I found that it works well and did not effect my viability/efficiency negatively.
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