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

[Pow Joshi]

> 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.

here are a few suggestions I can think of:

-try using polyA RNA for your hybridizations. I usually have gotten
some clean results with it.

-if increasing the temperation to 75º -80º might help

-check for probe degradation; partially degraded probe would give a
lot of nonspecific rRNA binding.

- I also had had some rather weird problems with binding when my
buffers were rather old .... the problems stopped once I made soem new
buffers.

best
pow










  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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