DIG Northerns

[Olivier Gandrillon]

Hi

We also tried DIG and 32P northerns side by side using RNA probes: 
sensitivity of 32P was, alas, about 5 times better than DIG (at the 
time, about 2 years ago: a new chemioluminbscent reagent was marketted 
later (CDP-star)). Most of DIG-related *problem* was due to a high 
background so that the film could not be exposed more than say 15 mn. 
So, if you could send me your protocol, I'd like to find out how you 
solved that background problem. What is sure, is that for genes that are 
sufficently expressed (GAPDH...), DIG Northern are just fine!

Olivier




Klaus Salger 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 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
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> 80333 Muenchen
> Germany
> 
> BioLinks: http://www.zi.biologie.uni-muenchen.de/~salger/salger.html