Denaturation of Streptavidin-Biotin bound dsDNA into ssDNA

Guo Wei Kua via methods%40net.bio.net (by gwkboy from gmail.com)
Thu Apr 8 23:24:49 EST 2010


Hi,

I have PCR products of about 70bp. One strand is biotinylated, because I
used biotinylated forward primers. Now, how do I isolate only this strand?
Do I denature first, then use beads to capture the ssDNA? If so, how to do
that? I am afraid that once I denature the dsDNA with heat, I won't be fast
enough to capture the ssDNA before reannealing.
Or do I capture the dsDNA first, then heat-denature the dsDNA so that the
non-biotin strand will fall off? The problems hereby is, again, reannealing
of the DNA. Second, heat-denature may even break the biotin-bead bond,
causing everything to be lost when I discard the sup? Help!!
Thanks.

PS: I prefer not to use high salt solutions, as the final purified ssDNA
should not hv salt if possible.


Regards,

gw


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