DIG Northerns

Klaus Salger salger at zi.biologie.uni-muenchen.de
Thu Jun 27 09:20:58 EST 1996


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 lowest
> 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.


Graham,

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.


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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