RNA Probe woes

Chutney devitta at uk.ac.birmingham
Wed Sep 21 06:13:10 EST 1994


In article <9409200024.AA25550 at cscgpo.anu.edu.au>,
Klaus.Matthaei at ANU.EDU.AU wrote:

> >In article <35jnec$lk9 at cc.tut.fi>, Jukka Luoma <bljulu at uta.fi> wrote:
> >>D.Greeve <DAEMON> (darreng at ccmar.csiro.au) wrote:
> >>: Hi netters.
> >>
> >>:    I am currently experiencing difficulties in producing P32 labeled RNA
> >>: probes from a "Bluescript KS+"  plasmid. I am currently using a kit
> >>marketed
> >>: by "Bresatec" (- an Australian company), designed to do this, but I am
> >>getting
> >>: almost no transcription using T7 and my plasmid. The kit control
appears to
> >>: work, however with identical conditions, my plasmid does not.
> >>
> >>: Any ideas on the matter would be much appreciated....
> >>
> >>: Darren Greeve.
> >>
> >>I have had similar problems and usually the cause of the promlems was too
> >>low plasmid concentration. Are you sure about your plasmid concentration?
> >>Your plasmid concentration should be about 0.1ug/ul in the final volume.
> >>
> >>Jukka
> >
> >
> >Another common source of problems is RNase in your plasmid prep.
> >
> >Carlisle
> 
> 
> Another common problem (a bit embarrassing but is has happened in our lab)
> is that the plasmid that you have cloned into isn't the one that you
> thought you had i.e KS+ vs KS-, pTZ19 vs pTZ18, so the promotor is pointing
> away from your insert etc etc
> 
> Cheers, Klaus
> 
> *************************************************************************
> Dr Klaus Matthaei                               |           |
> The John Curtin School of Medical Research      |  _--_|\   |
> The Australian National University              | /      \  |
> Snail mail: Canberra, ACT 0200, Australia       | \_.--._/<<|
> E-mail: Klaus.Matthaei at anu.edu.au               |       v   |
> Landline: 61 6 249 3782
>                         "Sometimes I'm the Louisville slugger"
>                         "Sometimes I'm the Ball"
>                                 Dire Straits
> *************************************************************************

There are a few possible explanations for this.
1> RN-ase in your plasmid prep, so try phenol:chloroform cleaning that and
make sure you include an RN-ase inhibitor like RNA-guard
2> When you linearise your plasmid the enzyme you use and the end it
leaves can also be crucial.  You are best to leave a 5' overhang.  A blunt
end may give problems and a 3' o/h is even more likely to give problems.
3>secondary structure may, as suggested above, be a problem but I have
never come across this.  Even probes that have a stable secondary
structure which makes them unsuitable for Ribonuclease protection assays
still transcribe well.  You could try linearising the plasmid with a
restriction site in the insert.  This would shorten the probe would would
reduce the stability of any 2ary structures formed. Please feel free to
Mail me with any queries as I and my colleagues have considerable RNA
experience.



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