Denaturation of Streptavidin-Biotin bound dsDNA into ssDNA

Guo Wei Kua via methods%40net.bio.net (by gwkboy from gmail.com)
Sun Apr 11 20:43:00 EST 2010


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 <dk from no.email.thankstospam.net> wrote:

> In article <mailman.446.1270818240.25217.methods from net.bio.net>, Guo Wei Kua
> <gwkboy from 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.
>
>
>
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