Electrophoresis of dsDNA and ssDNA

Glenn Dunshea via methods%40net.bio.net (by glenn.dunshea from gmail.com)
Fri Jan 2 02:25:59 EST 2009


I do SSCP analysis often and can confirm that with PCR product around 200bp,
the single stranded DNA runs much slower then double stranded. (I can send
you a pic if you like).

Here is a ballpark suggestion you may wish to try:

You may want to try denaturing the sample you wish to confirm is dsDNA and
see if it produces the same electrophoretic banding as a non denatured
sample.

Heat an aliquot at 95 degree's for a few minutes, take off and add one
volume of stop solution (95% formamide, 10mM NaOH, 0.25% bromo blue, 0.25%
xylene cyanol). Run this out in a PAGE gel (I am unsure of an appropriate
PAGE mix suitable for SSCP type things - I just use 1x pre-mixed MDE
solution but I'm sure you could find an appropriate PAGE mix in the
literature somewhere)  beside another aliquot of the same not denatured. If
they produce the same electrophoretic band pattern then the sample is single
stranded. If it is double stranded there will be additional bands closer to
the well in the denatured sample.

Short of a few details I know but potentially a start?



On Fri, Jan 2, 2009 at 8:31 AM, Paul J. Phelan <Paul.Phelan 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?
> 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?
>
> Thanks in advance,
>
> Paul
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>


More information about the Methods mailing list