TNT in vitro transcription/translation system

Paul Digard pd1 at mole.bio.cam.ac.uk
Fri Oct 10 16:39:03 EST 1997


Frank O. Fackelmayer wrote:
> 
> Hi all,
> We sometimes use in vitro translation to get protein synthesized from cDNA
> clones. In the last time, we´ve used the Promega TNT system for coupled in
> vitro  transcription/translation, which works very well. We have been
> using the T7-polymerase based system, and now I need a T3-polymerase
> system for only one or two reactions. I got plenty of the T7 system left,
> and I thought that adding T3 polymerase instead of the T7 polymerase
> should do the job (reaction conditions for the two polymerases are
> essentially the same in standard in vitro transcription assays). Has
> anybody tried that? Successfully? How many units of polymerase do you use?
> I really dont want to buy a complete T3 system for only one or two reactions.
> 
> Thanks,
> Frank

Hi

Rather than buying TNT kits at all, we use plain (and much cheaper)
retic from Promega as a base for the coupled transcription-translation
system described by Craig et al (ref below).  T3 substitutes fine in the
T7 optimised version, so I'd guess it would work OK in the commercial
TNT.  SP6 also works in the Craig system, but not quite as well in my
hands.

Hope this is useful

Cheers

Paul

Craig, D., Howell, M. T., Gibbs, C. L., Hunt, T. and Jackson, R. J.
(1992).  Plasmid cDNA-directed protein synthesis in a coupled in
vitro.transcription-translation system. Nucleic Acids Res. 20,
4987-4995, 1992
-- 

****************************************************************************


Dr Paul Digard
Division of Virology                    Tel 01223 336921
Department of Pathology                 Fax 01223 336926
University of Cambridge
Tennis Court Road
Cambridge CB2 1QB
UK



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