[Q] How to purify small PCR frg. ?

Michael Myers myersm at rockvax.rockefeller.edu
Tue Jul 25 11:26:38 EST 1995

In article <3ust1k$lvd at galactica.galactica.it>, Giorgio Spagnol
<spagnol at galactica.it> wrote:

> In article <950719.111158.19418 at macpost.lidac.liu.se> Sailesh
> Surapureddi, SaiSu at MCB.LiU.SE writes:
> > >Hi netters,
> > >
> > >recently a coworker of mine ran into a problem:
> > >She created a library with PCR using randomized primers,
> > >then she digested the fragment and want to ligate it into a vector.
> > >The complete PCR fragment is 96bp, after digestion she gets
> > >two fragments: 29 and 67bp. She separated them by gel
> > >electrophoresis on a 2.5% AG.
> > >The problem is now: How to purify the bigger fragment from the
> > >gel slice with a really *high* yield?
> > >She tried freeze-and-squeeze and Qiagen-gel-ex but got
> > >only very low yields.
> > >Any suggestions?
> I would try electroeluting them from polyacrilamide gels. However, I do
> not know what does it means for you *high* yeld.

Acrylamide gives the best resolution, obviously. Crush and soak usually
works for me. Another thing I've tried with success is the standard
2%NuSieve/1% normal agarose, excise the band, then purify DNA fragment
with Qiagen's product called QIAquick. Basically: the gel slice is
solublized in chaotropic salt, you spin through a filter device in
microfuge (DNA binds the filter), spin a wash solution through once, then
elute into 30 microliters of water or TE. Works great. NuSieve will
solubilize just fine.

Another nice agarose from FMC is MetaPhor. Slightly more tedious b/c must
set the gel at 4 degrees for 30 min before using, but it gives great
resolution. Haven't tested whether it can be dissolved with the QIAquick

Michael Myers
myersm at rockvax.rockefeller.edu

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