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During electroporation of bacterial cells (I work with Mycobacterium tuberculosis, but I think this applies to E. coli as well), sometimes I get sparking. I've read this is due to salts present, either in the DNA sample or the bacteria.

I use the Qiagen plasmid prep kit, which includes EB (Elution Buffer). This contains Tris-Cl. Could this be contributing to the sparking? Can you explain how this works? Does eluting with water instead of EB reduce chances of sparking?

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What media are the bacteria in during electroporation? –  GWW Oct 9 '12 at 4:24

2 Answers 2

In my experience the elution buffer does not matter (both for E.coli and Mycobacteria). I have heard this before from co-workers, but it usually is based on superstition or anecdotal evidence. I have compared water/EB elution and never saw a difference in sparking (in fact, I usually get slightly more colonies with EB).

When I get arcing (sparks), it's usually because I did not wash enough, have bubbles or have to many bacteria. Arcing is a collapse of resistance.

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Sparking occurs from having too many salts or possible not enough solution. After fine tuning my electroporation for very high amounts of DNA (16+ ug), what I've found to help are:

  • Make sure you are using the proper amount of liquid solution to transform. Too little will end up sparking
  • Clean up your DNA. I wash with twice the recommend amount of buffer PE, but I don't great evidence of how much the 2nd PE wash helps
  • Elute your DNA clean up with water
  • Drop dialyze your DNA. This is probably the most important step to prevent arcing
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