I was wondering why in transformation when calculating transformation efficiency do we divide by the supercoiled DNA
Transformation efficiency=Colonies/?g of supercoiled DNA.
Is the (bigger)recombinant plasmid DNA more supercoiled than the (smaller)non-recombinant plasmid DNA? Therefore, in that way we only calculate the recombinant plasmids in the denominator?

Thank you very much,

Ligated, nicked circular, linear and single-stranded DNA transform with much less efficiently than supercoiled DNA in an undigested plasmid - supercoiled DNA in this case gives you the maximum theoretical transformation efficiency. You can have denatured supercoiled plasmid DNA (that may be resistant to restriction enzyme digests) but it will still transform (contributing to ‘background’ in your efficiency calculations).

I seem to recall that there is no significant supercoiling of DNA until a circular molecule is at least 1kb in length. And I think an approx. 3kb plasmid has about 20 or so superhelical turns. In general large plasmids transform less efficiently than smaller ones, so while a larger plasmid may have more supercoils it is not necessarily going to transform at a higher efficiency. Added to that is the fact that your ligation reaction will be a mixture of ligated plasmid, un-digested DNA, etc. Hope this helps!

Last edited by Steve Lolait (22nd Apr 2011 06:44:47)

There should be no theoretical limit to the size of the plasmid being transformed, given that properly supercoiled plasmid molecules are reasonably compact structures.

Assuming that a 3kb and 50 kb plasmid have net neutrality effect upon introduction into a cell, it has more to do with the fact that the common unit of efficiency is based on a mass rather than number of moles. If you make sure you transform the same number of plasmid molecules, then you should see some consistency between the sizes (which is what I see when I work with my 50 kb+ plasmids - it just takes a bit more effort to prepare sufficient concentrations of supercoiled large plasmids!).

I can’t say that I have systematically compared the effects of plasmid size on transformation efficiency, rather I have a general impression using different-sized plasmid constructs containing ligated inserts expressed in CaCl2-treated (rather than electroporated) E.coli strains. Others say that they have made the comparison (on a molar basis) and concluded that the ‘size is important’, e.g., see - http://www.ncbi.nlm.nih.gov/pubmed/6345791

For other types of vectors (e.g., BAC, YAC) transformation efficiencies may also be affected by the size of the DNA, e.g., http://www.ncbi.nlm.nih.gov/pubmed/2045094
and may be strongly dependent on the bacterial strains used, as observed in some studies, e.g. - http://www.ncbi.nlm.nih.gov/pubmed/7596828

Last edited by Steve Lolait (25th Apr 2011 17:12:36)