I am trying to transform a plasmid that I prepared with the backbone, spacer, and repair templates (approx 14Kb) into a strain of S. aureus (ATCC 6538). I tried preparing electrocompetent cells using 0.5M sucrose (prepared in de-ionized water) and tried electroporation. However, I got a spark. I have also tried different settings for electroporation.
I was wondering if there is an alternative to electroporation in this case. Has anyone been successful with any other protocols?
Any help is appreciated.
Thank you.
If you get a spark during electroporation that usually means there is some salt in the mixture, normally from the DNA you are adding. You might try and clean your DNA to remove any excess salt and try again.
I don't think it's an S. aureus problem. If you just need a few clones and can't optimize electroporation, simply mix plasmid and bacteria and incubate, then plate. Possibly try some heat shock protocols from the E. coli people.
Most of the protocols for electrocompetent cells I've seen use 10% glycerol. Maybe you could use that instead of sucrose. Here it is an example of protocol for preparing electrocompetent cells and for their transformation: https://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells
Since your plasmid is big (over 10kb), I believe electroporation is still a better approach than heat-shock.
YOU CAN USE 10% GLYCEROL FOR ELECTROPORATION WITH 24V POWER. USE FRESHLY PREPARED MID LOG PHASE CULTURE.
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