T7 transcription of oligos
Claus Stefan Voertler
voertler at exmed1.gwdg.de
Tue Jun 18 16:05:34 EST 1996
have not seen these problems myself, but have heared of them. First of
all: from what you wrote I got the impression the resulting ladder
varies from experiment to experiment. If your oligos anneal in every
transcription the same way and all the components are identical, you
should always see the same products (evenso they might not be what you
want ;-) ). So maybe a problem in the set-up, annealing etc.?
Getting longer products then wanted could be caused by RNA itself,
which can be elongated by the enzyme (there was a paper by Picirilli
in Biochemistry a few years back .. although I do not recall if it
works for such long streches you discribe). On the other hand the top
band is roughly twice as big as your 30nt aimed product...sounds a
little bit lunatic, but maybe the template dimerizes?
The problem of a continous ladder up to the expected product of 30nt
points to problems the enzyme has allready on its way proceding
through the template - usually you see just abortion products (thats
what these band are) up to 9-10 nt, but then almost nothing up to full
length. So, T7 RNA Pol is not very well elongating here - you
mentioned that the transcript should be very U-rich ... but infact T7
RNA Pol just loves Gs in the template, especially in the beginning
directly after the promotor. So the problem could be the sequence
rather then the transcription conditions. To help the enzyme and avoid
an to early abortion you could incubate it at lower Temp (30 or even
lower). Try to make the enzyme feel as comfortable as possible - check
eg pH (also of NTPs!), Mg-conc. (4mM above total NTP-conc) etc.
The total yield of full length product depend on all the factors
above, but also on the initiation phase (first 10 nt polymerized) -
from my experience G is neeeded here much more then later on in the
Hope it helps a little, Stefan
PS: I don`t think its a electrophoresis problem if you run the gel as
hot as possible and with the usuall 7 or 8M urea
Paul Digard wrote:
> Hi All
> We've been trying to synthesise short (about 12 - 30 base) RNAs
> by in vitro transcription of annealed oligos ...
> we've been getting very poor
> results; basically a one base ladder up to the predicted length, but
> also beyond. Today's gel had a ladder up to 30 (the expected size,
> but barely more intense than any other product), but then up to more
> than 70 nucleotides.
> We've gel purified the oligos. The transcripts are meant to be
> devoid of secondary structure (very U rich in fact), and are heated
> to 90 C before being electrophoresed on pre-run polyacryl/urea gels..
Claus Stefan Voertler MPI fuer experimentelle Medizin
D- 37075 Goettingen
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