Why do probes stick to RNA markers?

Pamela Norton pamela.norton at mail.tju.edu
Fri Sep 29 15:15:51 EST 2000


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In article <oberstra-B90836.11342029092000 at news.hrz.uni-kassel.de>,
Juergen Oberstrass <oberstra at hrz.uni-kassel.de> wrote:

> > > Many, but not all, of our 32-P labelled probes stick to the marker lane
> > > on our northern blots. This happens with Sigma and Gibco BRL RNA ladders
> > > and the bands the probes hyb to don't correspond always to the marker
> > > bands.
> > > 
> > > Does anyone else get this, and can anyone give an explanation as to
> > > whats happening?
> > > 
> > > If you can help us out with this query e-mail me at
> > > jonathan.howarth at bbsrc.ac.uk
> > > 
> > > Thanks for any ideas
> > > 
> > > Jonathan
> > > 
> Do you hybridize with in vitro transcripts or with oligo-labelled DNA?
> In general the markers are produced by companies using cloned inserts.
> Because nearly all trancription vectors share common sequences around 
> the MCS the part of your probe containing MCS sequences fits to the 
> marker. There are several ways to avoid this signals (use PCR product as 
> template, use RNA as probe, cut exactly beside your insert etc.), but we 
> love them because they make the length estimation easier. 
> 
> juergen

I agree with Juergen's response, but in addition you need to consider
that a low level of crosshybridization can have a major effect due to
the high copy number of the RNA markers. However, it is puzzling that
you have bands that don't correspond to the marker bands. Maybe you
have a low level of contamination in your loading dyes or something? 

Pam






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