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

Sasha Chittka sasha.chittka at kcl.ac.uk
Fri Sep 8 05:13:04 EST 2006


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 



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