DIG Northerns

[Thibaud Le Mouel]

>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

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******** **       **     ** * Th. Le Mouel  <lemouel at antibes.inra.fr.>  *
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