HELP - isolation of PCR product from PAGE

Matthias Wahl matthias.wahl at gsf.de
Thu Jul 26 10:12:42 EST 2001


Krzysztof Wasowicz <wasowicz at moskit.uwm.edu.pl> wrote in message 

Hi Krzysztof,

I have no idea about possible inhibitory effects of any reagent you
use for silver staining, but EtBr also works very well for staining
polyacrylamide gels and very low amounts of DNA can be stained quite
well with SYBR green or SYBR gold.
I would assume your problem is that you elute together with your DNA
something inhibiting your Taq (unpolimerized polyacrylamide ?) - there
has been a BioTechniques paper (29:694-696) describing something like
that - I do not find the paper right now, but as far as I remember,
they showed that Tag is inhibited when using DNA eluted from
polyacrylamide gels (the longer you elute, the more severe the
inhibition). I think they suggested doing the elution only for a very
short time, but you better read their paper for the details.
You could also think of using some kit for purifying your DNA after
eluting the DNA.

Matthias


news:<3B5FE8C0.3CA2E80F at moskit.uwm.edu.pl>...
> Hi,
> I have a problem. I am doing PCR, then running PCR product in 4% PAGE to
> resolve DNA fragment to get different PCR products. Then I silver stain
> the gel and cut out bands (I use silvering method described in
> Biotechniques, 1994, Vol 17, No 5, page 915 - shortly: 3 mins in 10%
> EtOH+0.5% HAc, 5 mins in above+0.2% silver nitrate, rinse 1 min ddH2O, 5
> mins in 3% NaOH+0.5% formaldehyde, 10 mins ddH2O). I incubate cut out
> fragments of gel in elution buffer (essentially the same as regular
> 1xPCR buffer) at 95 Centigrades for 20 mins and use eluate directly for
> another PCR. To my surprise I get no PCR product even with specific PCR
> fragment which should be specifically amplified with the primers I use.
> Am I doing something wrong? Does anyone has a "foolproof" method of
> silver staining PAGE gel for subsequent fragment isolation? Any
> suggestion will be appreciated. Please, e-mail me.
> 
> Krzysztof (Chris) Wasowicz
> Dept of Animal Anatomy
> Univ. of Warmia & Mazury
> Olsztyn, Poland
> e-mail: wasowicz at uwm.edu.pl




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