in vitro transcription

Frank Lee sw12345 at hotmail.com
Mon Oct 18 18:12:09 EST 1999


There are possible two reasons. 1) RNase contamination. No matter how you
are careful, it is hard to avoid. You can add RNasin (RNase inhibitor from
Promega) or add RNasecure from Ambion (much less expensive than RNasin). I
prefer to use RNasecure.
2) Spot-like gel is caused by bad gel electrophoresis itself. Be sure to
prerun the gel and get rid of urea in the well just before you load your
sample, also, use a loading tip rather than a normal tip

Petra wrote:

> I am trying to get ribozyme RNA by doing in vitro transcription.
> Therefore I use freshly synthesized and non degraded, hybridised DNA
> oligos (I already tried different concentrations from 100 to 800ng); the
> T7 RNA polymerase, buffer and DTT are from GIBCO. For the transcription
> reaction fresh radioactive UTP was used. After mixing together all
> reaction compounds, I incubate at 37° for 1,5 hours (or more). Stop
> solution is 7 M Urea.
> Unfortunately, there are only spot-like, very small bands seen after
> developing the film, which I can´t use for elution.
> 4 weeks ago the same experiment (with 100ng oligo-DNA) brought positive
> results and very good bands. But this was not reproducable. Now it is
> really a problem for me, because I can´t start kinetic experiments
> without having ribozyme and substrate RNA of good quality.
>
> If there is somebody, who has a helpful suggestion, let me know.
>
> Thanks and greatings from Petra.




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