Guo Wei Kua <gwk...@gmail.com> writes: > > 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!! > > PS: I prefer not to use high salt solutions, as the final purified > ssDNA should not hv salt if possible.
I think this is the difficult approach. Why not create just the strand you want with linear amplfication using a single primer. Add just the biotinylated primer to a "PCR" reaction with large amounts of template DNA (perhaps a previous pcr with both primers, diluted significantly, say 100x). Do cycling as normal, but expect linear amplification with production of only the primed strand. Done, unless you really need to eliminate ALL of the reverse strand. Otherwise, purify with biotin. With this method you could probably work without biotinylated primers at much lower cost. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
