RNA Probe woes

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Mon Sep 19 19:22:00 EST 1994

>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
>>: by "Bresatec" (- an Australian company), designed to do this, but I am
>>: 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.
>Another common source of problems is RNase in your plasmid prep.

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

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