During electrophoresis I know that the leading band is the supercoiled DNA just because we have been told so (as it migrates fastest thru the gel) but is there an easy way to determine that it is? Or just accept that it's always the case when using all 3 conformations?

G'Day Lily:

I guess that this is a standard plasmid DNA prep. Carefully prepared supercoiled DNA can be almost pure and be run as a standard - it should be by far the major band with usually some ‘nicked’ (open circular) DNA and linear DNA if the DNA was harshly treated during preparation. I’m assuming of course that no dimers or oligomers have been formed. You could treat the prep (or the gel-extracted, predicted supercoiled DNA) with topisomerase which will convert it to relaxed, closed circular DNA and alter its migration in the gel. 

In general supercoiled DNA will migrate the fastest in agarose gels, but not always - e.g., denatured supercoiled DNA (i.e., slightly less compact DNA caused by something like excessive alkalinity during plasmid DNA preparation) can migrate faster. The order of mobility of different DNA conformations on agarose gel electrophoresis is (slowest first) - nicked open-circular * linear * relaxed, circular * supercoiled * “supercoiled denatured”.

Other ways to determine whether the leading band is supercoiled DNA? You could exploit the changes in mobility of supercoiled DNA compared to linear and ‘nicked’ DNA in the presence or absence of ethidium bromide, or compare the migration distances versus different agarose concentrations (gradient) in transverse pore gradient electrophoresis (TPGE). I don’t think either method is commonly used, and both are more tedious compared to running a single agarose gel. see refs (both available online) -

Oppenheim, A. Nucleic Acids Res 9: 6805-6812, 1981.
Wheeler et al., Electrophoresis 13: 403-406, 1992.

There is also a type of 2-dimensional electrophoresis (which is quite cool to pick up the degrees of supercoiling in topoisomers) and/or a sophisticated gel electrophoresis system called TGGE (temperature gradient gel electrophoresis) that can resolve supercoiled DNA, see - http://nar.oxfordjournals.org/content/2 … .pdf+html. Hardly routine stuff though!

Last edited by Steve Lolait (16th Aug 2011 19:52:52)