T7 transcribed RNA probes

Torsten Boerchers borcher at uni-muenster.de
Tue Jan 16 13:23:35 EST 1996


Torsten Boerchers <borcher at uni-muenster.de> wrote:
>Forwarded from rhubner at molbiol.ox.ac.uk:
>
>hello Torsten,
> although you don't give details about those multiple bands I would 
>suspect either premature termination or/and read-through (not completely 
>linearized... 
>who  can COMPLETELY linearize plasmids?) of the T7 pol beyond the RE >site 

>>"multiple" bands depending on run-on transcription intensity... 
>wouldn't cutting with a second RE help troubleshooting? but, what 
>then ? (i.e., how to stop such rolling-circles efficiently?). In the >case of premature termination I fear nothing special can be done... 
>oh yes, you might also have alternative initiation (change into other >RNA pol vector to check? can be predicted by seq analysis to some >extend...)
> Obviously _I_ too need to read/hear/experience more about T7 pol 
>transcription initiation/termination... any pointers to starting places 
>welcome  ;-) Especially, how good (bad) is T7 RNA pol with modified >bases as compared to pols...? Apologies, for sooo many question marks...
> Have a nice time,
> Roland
>
>+++++++_+++
>X-News: molbiol.ox.ac.uk bionet.cellbiol:3534

Dear Roland,

I agree that I was not specific enough. What we observe
(also with the control plasmid which Boehringer supplies)
is the occurrence of distinct (!) bands of multiple  [ 2, 3(?),
4 fold, i.e. larger] size. Of course we also have some lower 
Mw smear due to premature termination but not so much that 
it is a problem. I don´t see how run through on a partly
non linearized plasmid should give rise to a dimer. What I
understood is that under some circumstances (i.e. 3´overhang)
the T7 RNA Polymerase might change direction when it sits at 
the 3´end and begin to synthesize an RNA strand of opposite 
direction. 
I could not yet find out whether people think that this strand
is still linked to the first strand (thus explaining the
2fold size) or whether any (mixed?) weired double strand
would have a different apparent molecular weight.

P.S. We observe exactly the same behaviour with PCR products
as template for T7 Polymerase as well.

Regards
Torsten
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