Real-time PCR quantitation

Jo Lauber jla at gbf.de
Mon Mar 22 05:36:22 EST 1999

Hy David,

we´re working with the GeneAmp 5700 with SybrGreen for a few month.
First of all think of the price. 100 reactions are around 400 $. And there
is nearly no way to try your own buffers because PE don´t tell you the
concentration of SybrGreen and ROX used for internal control. Additionally
you have to buy special tubes and caps of course they have to be from PE.
Then you have to check the primers for optimal concentration ratios and for
the slope of the reaction to directly compare the gen of interest with a
housekeeping gen (this is the same with the LC). We´re doing it right now
for several cytokins and chemokins and it´s a lot of work and money.
PE is using their AmpliTaq Gold in the kits. This enzyme is really slow, in
some template/primer combinations using normal enzyms (eg from Promega or
Boehringer) you can see a product in the gel after 27 cycles. With the
Genamp you see the product on the screen after 26 cycles. So I´m asking
myself where is the detection limit of the GeneAmp?
I don´t know the LC-program, but the PE-program is really what in my
imagination is a beta. No drag and drop, no automatics, no way to save your
settings etc. so it is no fun to work with it. But one has to note that it
is the first windows program of PE, so may be one has to be patience.
I believe that the PrimerExpress is a good program. Unfortunately they don´t
tell you that it is a MAC-version. So no MAC no fun. The windows version
they told me we can expect in the year 2000.
Beside all the stuff above the computer they sell you with the machine is a
Dell 166 Mhz with a 15` monitor, really a joke but it works.

After all it works, and it works very reproducible (CV below 10%, also
because they use ROX). And it really shows you how you´re pipetting. So when
you have enough money for the reagents and enough time it is a good machine.

In the end I think every machine has its advantages and disadvantages, best
documented with the difference in sample number and speed. The GenAmp can do
96 samples in 1 1/2 hour, the LC 30(?) samples in 30 Min.
If there are further questions don´t hesitate to ask.


PS.: Anyone tested own buffers ? Anyone interested in exchange of primers
and protocols?

J. Lauber
Mucosal Immunity
Mascheroderweg 1
D-38124 Braunschweig
Tel.: 0049-(0)531-6181-306
Fax.: 0049-(0)531-6181-306
EMail: JLA at GBF.DE

David MacHugh schrieb:

> I would be interested in obtaining information about anyone's experience
> with either the Boehringer-Mannheim (Roche Molecular Biochemicals)
> LightCycler or the Applied Biosystems TaqMan system.
> Also, does anyone know if any other manufacturers are producing similar
> equipment for real-time PCR quantitation?
> Thanks in advance,
> David
> ____________________________________________________
>    David MacHugh, Ph.D.
>    Smurfit Institute of Genetics,
>    Trinity College,
>    Dublin 2.
>    Ireland.
>                           Phone: (353-1)-608-2719
>                           Fax:   (353-1)-679-8558
>                           Email: dmachugh at mail.tcd.ie
> _____________________________________________________

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