TNT kit with T7 RNA Pol

Jen Logan jlogan at genetics.utah.edu
Sat Mar 30 14:22:30 EST 1996


(1) PCR products:  We have followed a protocol using T7-based TnT on 
T7-tailed genomic PCR products to look for truncation mutations in 
disease genes of interest. (described in van der Luijt et al. (1994)  
Genomics 20, 1-4,  and references within).  Translation was greatly 
enhanced when we included a Kozak consensus sequence in the primer 
tail 3' to the T7 sequence,  and just 5' to the initiator methionine.  

Looking through my notes, I found the sequence I used:

5'-ggatcc-taatacgactcactata-ggaacagaccacc-atgg -3' 
																(T7)										(Kozak)   (met + g)

... then,  of course, the sequence complementary to template,  in 
frame. 

Lore has it that a g in the first position 3' to the initiator Met 
codon also enhances transcription.  Disclaimer:  there is no one Kozak 
consensus sequence that's written in stone;  you may find sequences in 
the literature that are different than this one. I am embarrassed to 
say that I don't have the reference for Kozak consensus sequences 
handy ... maybe someone can provide it?  All of this info comes from 
somebody else's hard work,  and I'd like to be able to give credit 
where it's due.

(2) Plasmids:  I have never linearized plasmid templates (pGEM-based),  
and have nevertheless gotten consistently good results. Linearization 
is recommended if you're transcribing in RNA in an uncoupled system,  
but not necessarily when doing TnT.  Promega claims that salt 
conditions may greatly affect production of your favorite protein;  if 
you're into kits,  they sell a test system that lets you try different 
conditions to optimize production of your protein.

(3) Extraneous info -- as usual,  be careful of nucleases,  they can 
ruin your day.  Also,  another plasmid vector,  called pCITE,  
contains a ribosome binding site; you can really crank up translation 
of your favorite protein using this vector.  Again,  I don't know 
where to get it,  though.  Can someone help?

Jen Logan



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