Replacement for retic lysate in Promega TNT kit

Stephen R. Lasky, Ph.D. Stephen_Lasky at brown.edu
Wed Jan 4 09:27:32 EST 1995


In article <3edvi7$asg at lyra.csx.cam.ac.uk>, bcek1 at mole.bio.cam.ac.uk
(Bernard Khoo (WCI)) wrote:

> I have recently run out of the reticulocyte lysate that was included with
> my TNT SP6 in vitro translation kit. I've phoned up technical support,
and they
> claim that the retic lysate is unique to the TNT system, and that ordinary
> retic lysate cannot substitute. Since the kit costs 305 pounds and 1ml of
> Promega nuclease-treated lysate costs 90 pounds, I am loath to spend all that
> money just to end up with an excess of the other reagents in the kit,
especially
> all the useless positive control reagents.
> 
> Does anybody have any experience with this situation? If I substitute
with, say,
> nuclease-treated retic lysate, will there be a loss of efficiency, and if so
> how much?
> 
> Replies to me by e-mail please. Thanks.
> 
> Bernard Khoo   Wellcome/CRC Institute, Cambridge, UK.
>                E-mail: bcek1 at cus.cam.ac.uk or @mole.bio.cam.ac.uk

Bernard:  This was posted a while ago, I haven't tried it but I think its
the info that you wanted.  SRL

In article <noren-150894145812 at 192.138.220.177>, noren at neb.com (Chris
Noren) wrote:

> In article <32e4d8$iml at network.ucsd.edu>, T. S. Pillay <tpillay at ucsd.edu>
> 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?
> > 
> 
> TnT system from Promega works great!! There's no reason why SP6 or T7 would

I agree, except that we found we could fund an additional post-doc when we
stopped sending regular payments off to Promega.

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.
(OK, don't think we pay post-docs $200/mo. The above line was hyperbole)

After conversations with a friend (Dr. VandePol) 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             15mM      1.5mM   7.5 ul 1M stock
rNTP's (4)         1mM      100uM    50 ul each of 10 mM stock
                                    42.5ul water

A typical reaction:

10ul retic lysate(*)
1 ul (1ug) T7 vector DNA(+)
1 ul amino acid mix, minus met
0.5ul 35S methionine
2ul 10x buffer above
0.5ul T7 RNA polymerase (25u)
5ul 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.

One caveat is that we have tested this buffer for use with T7 pol, and
have no idea if it works well for T3 or SP6.

*-- we now use the regular Met- lysate from Promega. It works as well as
the "2x" lysate from the TNT kit. In fact, we tricked the sales rep into
learning 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 note of thanks.

dennis

Dennis Templeton
Institute of Pathology
CWRU School of Medicine

-- 
*********************************************************************
Stephen R. Lasky, Ph.D.
Roger Williams Medical Center/Brown University
Phone: 401-456-6572       Fax: 401-456-6569       e-mail: Stephen_Lasky at brown.edu
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"The speed of a computer is inversly proportional to the legnth of time it has been on your desk."  Michael Yablonski, circa 1989
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