HELP - isolation of PCR product from PAGE

Errol E.S.Kwan at massey.ac.nz
Thu Jul 26 17:35:13 EST 2001


I used silver staining of PAGE for my AFLP's and when it came to
extracting the DNA from the bands I'd cut out, all I did was place the
gel in 50 microlitres of sterile water and left it overnight at 4
degrees C and then PCR amplified the next day and this worked fine.

On Thu, 26 Jul 2001 11:54:08 +0200, Krzysztof Wasowicz
<wasowicz at moskit.uwm.edu.pl> wrote:

>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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