Title: Why undigested plasmid lane should not be used to determine plasmid size? Post by: miharu on Sep 12, 2012 this is about gel electrophoresis, and on the worksheet i have there is this question Why undigested plasmid lane should not be used to determine plasmid size as opposed to the digested one in another lane of the gel ?
any help appreciated greatly Title: Why undigested plasmid lane should not be used to determine plasmid size? Post by: smachel on Sep 12, 2012 I think it is because the undigested plasmid would still be in a circular shape
Title: Why undigested plasmid lane should not be used to determine plasmid size? Post by: smackerish on Sep 12, 2012 Aha! That's an excellent question - your instructor is doing a good job. There are a couple of factors involved but it boils down mostly to structure. Running DNA across a gel involves the DNA fighting its way through a mesh-like physical barrier of interlocked proteins or the equivalent, right? The smaller pieces move faster than the bigger pieces because it's easier for them to find a pathway through the mesh. Now consider a plasmid. It's a circular piece of DNA, right? In terms of 'practical size', it's going to move though a gel differently when it is a loop than if it was cut and stretched out as a line. It's not even that simple, either, because you remember that DNA has secondary structure, etc., right? So the simple loop of a plasmid may be twisted up in the same way that you can take a wire loop and twist it so that it becomes 'super-coiled' and looks like a tightly-packed set of smaller loops. This will make it run differently from a normal loop, too.
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