TNT kit with T7 RNA Pol
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:
(T7) (Kozak) (met + g)
... then, of course, the sequence complementary to template, in
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?
More information about the Methods