TnT system in vitro translation

Stefan Gruenert (WCI) sg124 at mole.bio.cam.ac.uk
Mon Aug 15 05:08:01 EST 1994


Dear Netters,

in 14485 somebody wrote:

>I would like to use the Tnt system or any other in vitro translation
>system to make protein from a plasmid which has both the T7 promoter and
>the Sp6 promoter. Which is better?
>
>Also, would it be preferable to use Retic lysate or Wheat germ?
>
>Has anyone used the tRNA ascend system to make biotinylated proteins in
>vitro?

Anybody using the TnT ?
Shall I tell you what it is ?
Major expensive reticulocyte lysate with a bit of RNA polymerase and 
nucleotides added, which they won't tell anybody.

I don't know for sure of course, but check out the following reference:

 TI: PLASMID CDNA-DIRECTED PROTEIN-SYNTHESIS IN A COUPLED EUKARYOTIC
     INVITRO TRANSCRIPTION-TRANSLATION SYSTEM
 AU: CRAIG_D, HOWELL_MT, GIBBS_CL, HUNT_T, JACKSON_RJ
 JN: NUCLEIC ACIDS RESEARCH 1992 VOL.20 NO.19 PP.4987-4995

Was my old benchmate who got it up and running in the lab and it works fine
for everybody in the lab. We don't really know why either. All we do is 
adding 0.3 mM each NTP, additional MgCl2 (depending on the construct1.2 - 
3 mM), and a tiny bit of T7-Pol to the lysate. A ridiculous amount of DNA (even
miniprep DNA is fine, but take care of the salt, the major problem with the
system is that translation is quite fuzzy about high salt, and RNA 
polymerases love high salt, as we seem to need only little RNA we stay as 
close as poss to the retic optimum). 
For some reason the RNA you produce in retic is really efficiently translated, 
minor amounts of any plasmid DNA give you maximal yields of protein.
Initially we thought it would work only for IRES containing RNAs, but it 
turned out to work fine for just any bog standard RNA as well. We use it 
lots for screening in frame deletions, but others don't even bother 
making RNA anymore.

As to SP6 or T7, we never tried SP6, but there is no reason why not, make 
sure you get the cloned stuff, which is really efficient

we also nerver tried wheat germ, but I wouldn't 
recommend it, WGE is usually a bit more temperamental and less efficient,
and I never biotinylated anything in my life,

Therefore, stuff the kits, and use your ownm,
hope it'll work for all of you,

Stef
________________________________________________________________________

Stefan Grunert                   _____/     |        ____/   |  /     /
Wellcome/CRC                    /         / |       /        | /     /
Cambridge/UK                   ____/     /  |     ___  /      /    _/
                              /         /__ |         /      /
SG124 at mole.bio.cam.ac.uk    ______/   _/   _|   _____/     _/    _/
_________________________________________________________________________  




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