[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
kit.

Michael Myers
myersm at rockvax.rockefeller.edu



More information about the Methods mailing list