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Need In vitro transcription vector and info

Steven Joseph Szarka sjszarka at acs.ucalgary.ca
Sun Aug 8 15:18:58 EST 1993


 
Hi Net:
 
 
We are planning to produce polyadenalated transcripts in vitro. 
For this purpose we have purchased the pSP64PolyA vector from Promega.
This vector contains a 30nt polyA tract at one end of the mcs.
The RE site following the polyA site for linearization is an EcoRI site.
Unfortunately, one of our coding sequences contains an EcoRI sequence.
 
Does anyone have a vector for in vitro transcription containing a
polyA tract of at least 30 As, with a rare RE site for linearization
(eg. NotI or PmeI).
 Ideally, the vector should contain a unique KpnI
and/or blunt cloning site in front of the polyA sequence.
Thanks in advance for any help.
 
 
Now, some more general questions about in vitro transcription:
 
1) How close to the polyA sequence must the linearization site
be? Is there a maximum distance?
 
2) Will the transcript extend around the vector if the
linearization RE site is  allowed to reanneal? 
If yes, then is the solution to use
a blunt site or end filled cohesive site for linearization? 
 
3) If I am testing the effect of several different coding
sequences, should they all be cloned into and transcribed 
from the same vector? (For consistency).
 
 
Thanks again for any help
 
Steve Szarka
University of Calgary
sjszarka at acs.ucalgary.ca
 
 
 



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