Oligo probes and Northerns

Garry P. Larson glarson at ccmail.llu.edu
Wed Sep 27 15:20:18 EST 1995

In article <44bno1$6nv at mercury.hgmp.mrc.ac.uk>, smjones at hgmp.mrc.ac.uk (Mr.
S.M. Jones) wrote:

> I am currently trying to establish the validity of a 37mer probe for insitus. To do this I need to hybridise it (radio labelled) to a Northern blot. 
> Ive tried the protocol I normally use for cDNA probes (200-500 bp) and fiddled with hybridisation temps (I have reduced it but not increased), wash stringency etc but I cannot get anything on the autorad.
> I know the RNA is on the blot as I run the gel with ethidium and this transfers during the blot.
> Am I just being unlucky or is there something else I could try
> Simon.

Is the RNA transferring efficiently?  Load some hot marker onto the gel and
check its transfer efficiency.
Is the RNA target you're screening for degraded?  Load a serial dilution of
some DNA containing the target sequence at concentrations similar to what
you expect for your target RNA.  They should light up.
Also keep the ionic strength at app. 0.9M Na+ (corresponds to 6X SSC or
SSPE)  If I'm not mistaken the hyb. solution for cDNA probes is NOT 6X SSPE
(or 6X SSC), its lower.
You might want to try lowering the hyb temp. also and apply stringency
during the subsequent washings.


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