Homemade Transcription/Translation (TNT) system

Mark Guiltinan Mark_Guiltinan at AGCS.CAS.PSU.EDU
Wed Aug 11 16:40:08 EST 1993


We too seek to avoid coporate price gouging on the Promega TnT system, which
comes with too little T7 polymerase than needed to use all of the retic lysate.
 We have found that you can use T7 polymerase at the lower unit per microliter
concetration available from Promega seperatly, saving lots of $s in buying new
kits.  Thanks for the magic buffer recipe.

Mark Guiltinan
Linda Miller
Penn State Biotechnology Institute
mjg at psupen.psu.edu
_______________________________________________________________________________
From: djt2 at po.CWRU.Edu on Wed, Aug 11, 1993 2:28 PM
Subject: Homemade Transcription/Translation (TNT) system
To: methods-and-reagents at net.bio.net


This is a re-post of a protocol I posted a while ago.  A reader pointed out
a mistake in one of the recipes (the final concentrations were right). This
is the corrected 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 10x stock
======================================================================
Tris.Cl pH 8.0	500 mM		50 mM		50 ul 1M stock
MgCl2		15mM		1.5mM		1.5 ul 1M stock
rNTP's (4)	1mM		100uM		5 ul each of 10 mM stock
						8.5ul water
						total volume 100 ul

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

*-- we are still using the retic lysate from the Promega kit.  It is
possible that this lysate is more concentrated than their other lysate, but
I don't think so. In fact, the rep said that I could substitute Promega's 
regular lysate for the TNT lysate for the T7, (but not SP6) without alteration.

+ -- 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 note of thanks.

Thanks too to my post doc Jan Wolfman for testing this recipe out.
 





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