in vitro transcription

tfitzwater at gilead.com tfitzwater at gilead.com
Mon Oct 18 14:02:30 EST 1999


>Petra (petrako at gmx.de)
>Mon, 18 Oct 1999 16:00:31 +0100

>Date: Mon, 18 Oct 1999 16:00:31 +0100
>I am trying to get ribozyme RNA by doing in vitro transcription.
>Therefore I use freshly synthesized and non degraded, hybridized 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.

Check Ambion's MegaShortScript protocol at
http://www.ambion.com/techlib/index.html

I suggest a 500-1000 ng/mL range for small transcription templates.

High yields can be obtained from Promega's T7 RiboMax buffer.
Alternatively, add PEG8000 to a final concentration of 4% to the buffer
system you are using.  Dissolve the PEG in boiling water and let cool
before adding the remaining buffer components.
I also recommend that you add yeast inorganic pyrophosphatase from Sigma
catalog number I1890.  To prepare a 10x stock, resuspend the lyophilized
material to a final concentration of 0.1 units/uL in pre-chilled 10 mM
HEPES-KOH, pH 7.5, 6 mM MgCl2 and 50% (v/v) enzyme grade glycerol.  Store
at -20°C.  To prepare 10 mL of PPase storage buffer, mix 100 uL of 1 M
HEPES-KOH, pH 7.5, 60 uL of 1 M MgCl2 and 6.25 mL of 80% (v/v) enzyme grade
glycerol with 3.6 mL of Type I water.  80% glycerol is easier to pipette
than 100% glycerol.

Promega, Fermentas and Ambion all offer high concentration T7 RNA
polymerase.  This is necessary for the number of initiation events required
for transcription of small templates.

In a 4-6 hour incubation, you should generate 3-10 nmoles (depending on the
sequence of your template at positions +1 to +15) from a 500 uL
transcription reaction if the NTP concentration is 2 nM each.   10 uL of
labeled UTP added to the reaction will still generate well-labeled RNA.

Tim Fitzwater
Gilead Sciences




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