problems with probe binding to 28S and 18S RNA only on Northerns

[Michael Sullivan]

What type of probe are you using. I ask because with some riboprobes  
I have seen this sort of thing-- very strong hybridization to  
ribosomal bands that can't be washed off. I've never seen a similar  
problem with DNA probes. That said, I overcame the riboprobe problem  
by increasing the stringency of the hybridization condition to be  
very close to the predicted Tm. This fixed the problem I was having  
with that particular probe.

Hope this helps.

Mike Sullivan



On Sep 8, 2006, at 5:13 AM, Sasha Chittka wrote:

> Hello,
>
> i have had a very peculiar problem lately with the Northerns - i  
> get the binding of the probe to 28S and 18S RNA only and no  
> specific signal.  I have never experienced this before.  The method  
> i used is the following:
>
> Total RNA was isolated by Trizol method and separated using a  
> formaldehyde containing gel.  The RNA looked beautiful judging by  
> 28S and 18S bands.  The transfer was onto charged Nylon membranes  
> from Amersham and was controlled by observing the 28S and 18S bands  
> under UV light.  The hybridisation was run at 65oC in the Church  
> buffer (7%SDS, 0.5M Na phosphate buffer pH7.2, 1mM EDTA and 1% 
> BSA) .  I washed with 40mM Na phosphate buffer, 10mM EDTA, 1%SDS   
> at65oC and then with 0.5xSSC, 0.1%SDS at 68oC with many changes.   
> The result was, that there was no background on the membrane, but  
> everything bound to the 28S and 18S bands and i saw no specific  
> signal at all.
> Has anyone had this problem and has any idea how to battle it?   
> please, let me know if you have some thoughts.  thanks a lot and  
> best regards,
>
> Sasha
>
> Alexandra Chittka, PhD
> MRC Centre for Developmenta Neurobiology
> 4th Floor New Hunt's House
> King's College London - Guy's Campus
> London Bridge
> London SE1 1UL
>
> Tel: +44-(0)207-848-6532
> Fax: +44-(0)207-848-6550 or 6798
> e-mail:  sasha.chittka at kcl.ac.uk
> _______________________________________________
> Methods mailing list
> Methods at net.bio.net
> http://www.bio.net/biomail/listinfo/methods

---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)