T7 transcription of oligos

Claus Stefan Voertler voertler at exmed1.gwdg.de
Tue Jun 18 16:05:34 EST 1996


Hi Paul,

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 
template. 

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 ...

 (SNIP)
> 
> 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..
 
  (SNIP)

__________________________________________________________

Claus Stefan Voertler     MPI fuer experimentelle Medizin
                          Hermann-Rein-Str. 3
                          D- 37075 Goettingen
                          Germany
__________________________________________________________

-- 
œ7



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