non specific bining of DIG probes

Mathias Holpert mholper at gwdg.de
Tue Jul 23 08:01:31 EST 2002



Carol Chihara schrieb:

> I am blotting cut plasmids with inserts and electroblotting to nylon
> membrane (+). The blots are fine, but the DIG labeled probe binds to
> all the bands including MW markers.  I have tried three different
> probes (two tailed oligos and one random primed cDNA), they all do
> the same thing. I have upped the stringency washing at 68deg in .15X
> SSC and still everything picks up the label.  Could this be due to
> the electroblotting?  I have been in touch with BM but they didn't
> suggest anything helpful, i.e,. I have decreased the DNA on the gel
> and the conc of probe, still the same problem.
> Thanks for any help.
> --
>
>   Dr.C.
> ---

Did you purify your DIG-labeled probe (run it through a gel if you made
it by PCR) after producing it? Maybe there are still some labeled
nucleotides around...

Mathias




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