Hi, I have actually trying to denature the dsDNA by heat. I don't get any DNA yield if I do that, i suspect that the strapt-biotin bond is broken tru the heat. So, I found a paper mentioning denaturing the dsDNA without heat. They actually used ammonium hydroxide and incubate at room temperature for 2 min. The dsDNA will become ssDNA, while the strep-biotin bond is only to a small extend broken. This should do the trick, doesn't it?
Thx GW On Sat, Apr 10, 2010 at 9:42 AM, DK <d...@no.email.thankstospam.net> wrote: > In article <mailman.446.1270818240.25217.meth...@net.bio.net>, Guo Wei Kua > <gwk...@gmail.com> wrote: > >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. > > I've never had to do what you want but did many things tangentially > related. Here is what I'd do if I were you: > > 1. Bind the PCR product to streptavidin sepharose. Wash well, alternating > high and low salt (to remove everything else). > 2. [Almost] boil the whole thing - say, couple minutes at >95C in the > presense of high enough salt (> 200 mM NaCl). > 3. Quickly cool in liquid nitrogen (prevents reannealing). > 4. Quickly melt at 95C by adding boiling buffer and wash several times > with very hot buffer, again, keep salt ~ 200 mM. > 5. Wash with whatever salt you want at any temperature. > 6. Boiling in 1% SDS in low salt will elute biotinylated ssDNA. > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
