T7 transcribed RNA probes

S.Bartoszewski S.Bartoszewski
Wed Jan 17 00:36:09 EST 1996

In article <4dgqf7$10g0 at majestix.uni-muenster.de> Torsten Boerchers <borcher at uni-muenster.de> writes:
>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 
>>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 
>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.
>                          Torsten Boerchers                            /
>    _/  _/_/_/ _/_/_/    Institute of Chemical- and Biochemical Sensor/
>   _/  _/     _/   _/   Research (Molecular Biology Group)           /
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>_/  _/_/_/ _/_/_/_/  E-Mail: borcher at uni-muenster.de              /
I haven't seen the original article, so I don't know what the size of
your bands is.  T7 polymerase is known to carry out a reaction of RNA
replication.  The polymerase is probably always contaminated with some peculiar
RNAs (about 70 bases long) and if no good template is delivered these
RNAs will be replicated and some will be multimerized.  You may check
papers by Konarska and Sharp: Cell 57, p423-431 and Cell 63, p609-618.
Cheers, Slawek.

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