HELP - isolation of PCR product from PAGE

Krzysztof Wasowicz wasowicz at moskit.uwm.edu.pl
Thu Jul 26 04:54:08 EST 2001


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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