Northern non specific ?

smoothies channels at sghms.ac.uk
Wed Nov 11 07:09:45 EST 1998


>
>
> Yes, Yes, Yes, this seems to be a common, frequently encountered
> PROBLEM for northerns. I am sure almost anyone in the field has seen
> nonspecific binding to 18S and 28S ribosomal RNAs, and even so
> in published papers.
> This is mostly because riboprobes are "STICKY"!
>
> I would recommend at least two strategies:
>
> * try to use shortest hyb time, and highest hyb temp
>   (2-3 hrs, ~70degC, rapid hyb cocktail cont SDS)
>   Not only washing at high stringency is important!
>
> * if probe is prepared by T3/T7 polymerase include
>   a short unlabelled competitor RNA (in excess) derived by the same
>   strategy (run off) from nonrec plasmid (traditionally pBS)
>
> ...this is what my experience tells me...hope this helps,
>
> Mart
>

Thank you Mart for your advice & experience.
But I am running mRNA on my gels (OK they might still contain a bit of
rRNA)
and I use DIG Easy Hyb buffer (seems to contain SDS) overnight at 68
degrees C.
My final wash is 0.1xSSC,0.1%SDS at 68 degrees C, for 15 minutes twice.
My supervisor, who is not a molecular biologist, says this must be non
specific binding -
I get no background on the membrane but the "positive" area is a bit
smeary and seems to
contain a band - I say this is because I loaded too much mRNA (5ug) but
he doesn't
beleive me...
In the same lane, running higher I get a nice clean positive band which
could rule out that I
have mRNA degradation.
Anyway, I have repeated the experiment with various amount of mRNA now.

Claire Fenech
PhD Student
Pharmacology - SGHMS, London




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