eluation of small DNA fragments from gel

bench_monkey bench_monkey at yahoo.com
Tue May 9 21:09:08 EST 2000


try electroelution. it works equal well for polyacrylamide and agarose
samples.  you will still need to remove residual polyacrylamide for
downstream apps.  hplc gives the best results although it requires a bit
more dna and a few extra minutes.  you will be happy with the results
though.

<grey at em.uni-frankfurt.de> wrote in message
news:3917d17c at nntp.server.uni-frankfurt.de...
> Matthias Wahl <matthias.wahl at gsf.de> wrote:
>
> >Could anyone tell me a good method for eluting small DNA fragments
> >(20-30bp) from Polyacrylamide? It could also be a method for eluting
> >from Agarose (Metaphor etc). My problem is, that most methods use
> >temperatures, at which such small fragments will melt.
>
> >I currently use 12% PAGE, and elute just by incubating together with
> >buffer for >2h at 37=B0C and afterwards just filter away the
> >polyacrylamide. My problem is that I only have a very low efficiency in
> >a subsequent ligation step. Propably I carry over some contamination
> >from the polyacrylamide. Is this likely that remaining polyacrylamide is
> >inhibiting ligation reactions, or do I just have some impure reagents
> >for making the gel.
>
> >I also looked for some commertial kits for purifying the eluted DNA but
> >they are not suitable fur fragments of this size.
>
> >Thanks in advance
>
> >Matthias Wahl
> Matthias Wahl <matthias.wahl at gsf.de> wrote:
>
> >Could anyone tell me a good method for eluting small DNA fragments
> >(20-30bp) from Polyacrylamide? It could also be a method for eluting
> >from Agarose (Metaphor etc). My problem is, that most methods use
> >temperatures, at which such small fragments will melt.
>
> Hi Matthias,
>
> Try this one (see below). For small fragments, its working great.
> No heat required. The filter disks can be obtained from Schleicher &
> Schuell (normally used for antibiotics). Good luck
> --------------------------------------------------------------------------
-----------------------------
> Grey M, Brendel M
> Curr Genet 1992 Jul;22(1):83-4
> "Rapid and simple isolation of DNA from agarose gels"
>
>
>






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