Electrophoresis of dsDNA and ssDNA

ddloeb from gmail.com via methods%40net.bio.net (by ddloeb from gmail.com)
Fri Jan 2 11:50:40 EST 2009


On Jan 1, 5:31 pm, "Paul J. Phelan" <Paul.Phe... from tufts.edu> wrote:
> I have a simple question that so far does not seem to have a simple answer.
> I need to confirm that a 60-mer dsDNA oligo is double-stranded.
> This is poly-dA/dT DNA.  Does anyone have a straightforward 1D
> electrophoresis protocol that will separate single-stranded and
> double-stranded forms of an oligonucleotide in the same range as a
> 60-mer oligo?

Run them out on a 10% native PAGE gel in 1XTBE.  Run the gel at 17oC.
This procedure was used to separate fully-folded tRNA from partially
unfolded forms.

> I have already been surprised to learn that in 7 M urea PAGE, ssDNA
> will run SLOWER than dsDNA.  Does anyone have experience with this?

In 7M urea, it is extremely unlikely that you have any dsDNA -
especially for something that is AT-rich.

Dan


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