TNT coupled T7 reactions (homemade)

[Dennis J. Templeton]

A recent discussion centered on the relative merits of the coupled
Transcription 'n' Translation system of Promega. I agree with the comments
of others, that this is a reliable procedure preferable to the uncoupled
reaction, except at the level of price gouging. I am reposting our recipe
for the homemade version, which in our last test worked better (25% more
yield) than the promega version.

We are satisfied users of the TNT system from Promega; the one-tube
translation and transcription system that allows you to simply add a T7
plasmid into a reticulocyte lysate and get radiolabelled plasmid out.  That
is, we are satisfied *except* for paying 3.5x the price of the standard
translation system.  Promega sends you about 50% more retic lysate than you
can use with the tiny aliquot of the magic buffer that has ribonucleotide
triphosphates and whatever other goodies are needed to allow the T7
polymerase to work in the retic lysate.

In an attempt to combat corporate secrecy and price gouging, we have sought
to duplicate the conditions used in their "secret transcription buffer".
The buffer below proved just slightly superior to the buffer supplied with
the kit, at a savings of over $200 for about a month's supply at our rate
of usage.

After conversations with a friend (Dr. VandePol of NIH) we made up the
following buffer:

																	10x conc			1x conc	 									Recipe for 500 ul of	10x
stock
========================================================================
Tris.Cl pH 8.0					500 mM				50 mM														250 ul 1M stock
MgCl2															15 mM			1.5 mM														7.5 ul 1M stock
rNTP's (4)											1 mM			100	uM						 								50 ul each of 10 mM stock
																																															42.5 ul water

A typical reaction:

10ul retic lysate(*)
1 ul (1ug) T7 vector DNA(+)
0.5ul 35S methionine
2ul 10x buffer above
0.5ul T7 RNA polymerase (25u)
6ul water

Reaction is for 30 minutes at 30 degrees. We have scaled this down to as
little as 5 ul (drying the DNA down first) to screen minipreps by
expression.

*-- we now use the regular Met- lysate from Promega. It works as well as
the "2x" lysate from the TNT kit. In fact, we learned through the sales rep
from  the tech rep that it is in fact the same product. The tech rep said
that the Cys- lysate wouldn't substitute. I don't know why.

+ -- We use the vector pTM1 from Bernie Moss (NIH) that has a 5' leader
that enhances translation of uncapped messages, and a T7 terminator as
well.  With this vector no pre-digestion of the plasmid is required. 
Novagen markets a similar vector (pCITE).

In fairness, Promega deserves credit for developing this system, and if
they priced their reagents consistent with the labor involved in making
this reagent I would gladly pay it.  In fact, I hope to continue buying the
vanilla-flavored retic lysate from them as a small(er) note of thanks.




-- 
Dennis J. Templeton
CWRU School of Medicine