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From my limited knowledge of science, I know transformation can be one of the hardest step in cloning, and that a large amount of research/trial and error has been done to improve on this step. I've heard electroporation is the most efficient technique, though I have no reference, and of course the cell, media and duration all have vital parts to play in the process.

However, I've never heard of supercoiling as an important factor. Size of the plasmid plays a large part in transformation, so intuitively it makes me think supercoiling the plasmid prior to transformation would improve the efficiency. I thought transformation was simply DNA diffusing through holes, but are parts of the plasmid required to bind/interact with the host?

Why aren't we suggested we supercoil prior to transformation? I have several thoughts but I'm just guessing:

  • Is it because gyrase can disrupt the integrity of DNA?
  • Or is it that it's just an expensive enzyme and the gain isn't worth it?
  • Or perhaps supercoiling reduces efficiency of subsequent plasmid preps/transcription (though wouldn't the host's natural enzymes supercoil anyway?)
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    $\begingroup$ Two comments on this from my lab experience with transforming bacteria: Making cells competent (I usually use chemical competent cells since we have no electroporator in our lab) is not a hard thing to do. Transformation effeciency isn't usually a problem either (with good competent cells you easily get 5x10^8 transformants (and more) per ug of DNA. So my guess, why nobody bothers to use an additional step (besides the time and money it costs for the enzyme), is that the methods we have are simply good enough. $\endgroup$ – Chris Feb 1 '14 at 20:46
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Here is a report that shows significant increase in transformation efficiency for large plasmids (transformation was by electroporation. However the paper has been cited fewer than ten times so clearly the technique did not catch on.

Zhixing, Y and Nahon, J-L (1995) DNA gyrase improves DNA transformation of E.coli cells with large recombinant plasmids. Nucleic Acids Res. 23:3353-3354

enter image description here

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  • $\begingroup$ Thank you, interesting to know in case of any problems with transformations that this might actually work! $\endgroup$ – Richard Webb Feb 2 '14 at 2:49
  • $\begingroup$ The paper predates bluelight and sybr safe, which improve the efficiency by several orders of magnitude, so they were definitely ahead of their times. The reason why nobody does it is that from what I can tell they use Micrococcus luteus gyrase because they had it. Sigma sells Eco gyrase but at £500 I am not sure who would try it —I myself am working with epPCR libraries with a big plasmid yet I wouldn't spend that much. $\endgroup$ – Matteo Ferla Oct 2 '16 at 11:03
  • $\begingroup$ Correction. (NEB)[neb.com/products/m0306-dna-gyrase-e-coli] sells it for USD70 —for 100 0.5 µg rxns. That makes it worth a shot... $\endgroup$ – Matteo Ferla Oct 2 '16 at 12:31

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