DIG Northerns

Klaus Salger salger at zi.biologie.uni-muenchen.de
Fri Jun 28 11:55:39 EST 1996


Thibaud Le Mouel wrote:
> 
> >Graham Atherton wrote:
> >>
> >> Has anyone tried the DIG system for Northern hybridisation in direct
> >> comparison with 'good old' 32P labelled probes - which picks up the lowes=
> t
> >> amount of target RNA with the least background? Is there a better
> >> non-radioactive
> >> system than DIG? Boehringer clain comparable results with 32P but I hear
> >> differently.
> >
> >Klaus Salger reply:
> >
> >>I did a direct comparison of radioactive and DIG hybridizations. The
> >>radioactive one was done with formamide buffers, the DIG northern was
> >>hybridized using a phosphate buffer with high SDS.
> >>Background was no problem with both of them. Both were equally
> >>sensitive. So the smallest amount of RNA that gave a band was the same
> >>with both but while the radioactive blot gave big blobs with the strong
> >>signals the DIG blot produced rather sharp bands that didn't look as
> >>"impressive" as the radioactive signals.
> >>I think the dynamic range is much smaller with chemiluminescence (on
> >>film)!
> >>So if you just want to SEE the band, the DIG system is fine but if you
> >>want to quantitate the signal strength the radioactive system is better
> >>(especially with a phosphoimager).
> >>
> >>If you want to see the film I could send you a scanned image by e-mail.
> >
> >
> I've tried DIG hybridization using PCR for the probe and formamide and high
> SDS at 42=B0C for hybridization. The sensitivity was nearly the same with P3=
> 2
> BUT... I have a lot of Background so I'm interrest if Klaus can give me his
> protocol and send me his film.
> 
> Thanks a lot. Thib


I put the DIG protocol and the scanned image on my web page (URL: see
below). So everybody can have a look.
Questions, suggestions, comments etc. are most welcome.

Cheers
  Klaus

-- 
Klaus Salger                phone : +49 (0)89 5902 -502
Zoologisches Institut       FAX   :                -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
Luisenstr. 14               
80333 Muenchen
Germany

BioLinks: http://www.zi.biologie.uni-muenchen.de/~salger/salger.html



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