denatured closed circular plamids, two separated rings or catenated rings? - (Jul/19/2006 )
i want to check the quality of my crosslinked supercoiled plasmids, i wonder that under denaturing condition, supercoiled plasmid will formed two separated single strand rings, or the two single strand rings will interweave or catenate with each other?
The two strands stay together, since the plasmid is so tightly coiled and wound up in itself. This is a property that is useful for extracting plasmids under alkaline conditions--large genomic DNA gets denatured and tangled into the mess of cell debris in lysate, while plasmids remain largely intact and the ss plasmid DNA can be allowed to reanneal with no problem.
Thank you, Meres, that helps a lot.
So, it is impossible to tell the difference between crosslinked supercoiled plasmid and non-crosslinked supercoiled plasmid by running a denaturing agarose gel.
If by crosslinked you mean annealed, then yes, I don't think they'd look any different. There's no way to see it on a gel since the only difference is disrupted h-bonds, not the positions or association of the larger circular molecules. If you treated the plasmid really roughly you might bust it up, but then it wouldn't be supercoiled anymore; you will see loose circular and linear plasmid caused by nicks (I'm sure you know this).
by crosslinking I mean using a chemical (TMP, derivative of psoralen) to covalently binding the two strands at one special site of each supercoiled plasmid.
Aha, I see now--sorry for my braincramp. So still, no--if the plasmid remains supercoiled, you will not tell the difference between the two. The strands will stay associated whether they are covalently linked or not.