perkin elmer vs light cycler

Weining, Song WeininS at prose.dpi.qld.gov.au
Tue Aug 24 06:03:33 EST 1999


There is some major difference between PE cyclers themselves in the
heat/cooling mechanism. Consequently, the PCR profiles often need to be
optimised. The difference between the light cycler and other PCR machines is
even greater. 
It will take too long to list all the details here and I have been working
on a book to discuss it.
With the light cycler, you have to not only optimise the amplification
conditions, essentially annealing temperatures, but also include BSA in the
reaction buffer which is usually supplied in a optimising kit with the
machine. Optimisation, even with the gradient machines, is one the most
frustrating tasks in the lab. Do it with a light cycler or a capillary
cycler (from the same company)? I wish you all the luck in the world. I have
to emphasise here I don't mean Idaho makes bad machines. Actually their
machines could be fast and wonderful if you begin your PCR with them but not
the other around. 
The moral of the story, I believe, is never to transfer PCR profiles from
one machine to another unless you are prepared for the optimisation task.
S. Weining, PhD
Molecular Biologist
Leslie Research Centre
13 Holberton Street
PO Box 2282,
Toowoomba, QLD 4350
Australia

Phone: 61-7-46398880
Fax: 61-7-46398800
Email: weinins at dpi.qld.gov.au

Arul Jayaraman (jayarama at helix.mgh.harvard.edu
<mailto:jayarama at helix.mgh.harvard.edu>)
Tue, 3 Aug 1999 16:40:53 -0400 
*	Messages sorted by: [ date ] <date.html>[ thread ] <index.html>[
subject ] <subject.html>[ author ] <author.html> 
*	Next message: 915613 at candseek.com: "JOBOP Molecular Biology
Scientist" <0090.html> 
*	Previous message: Achim Recktenwald, PhD: "Re: protease
purification" <0088.html> 
*	Next in thread: Thomas Pfitzner: "Re: perkin elmer vs light cycler"
<0096.html> 
*	Reply: Thomas Pfitzner: "Re: perkin elmer vs light cycler"
<0096.html> 

To: methods at net.bio.net <mailto:methods at net.bio.net>
From: Arul Jayaraman <jayarama at helix.mgh.harvard.edu
<mailto:jayarama at helix.mgh.harvard.edu>>
Subject: perkin elmer vs light cycler
Date: Tue, 3 Aug 1999 16:40:53 -0400
hi
our lab has invested in the light cycler from idaho technology and we are
having some trouble with it. we had previously done rt-pcr of few
transcripts using the thermal cycler from perkin elmer and we got lot of
product. however, when i do the rt in a thermal cycler and the pcr in the
light cycler, i do not get any specific product. the reason i do the rt
and pcr separately is because promega's rt-pcr kit won't work with the
light cycler (it uses MgSO4 as opposed to MgCl2 with the light cycler).
the conditions are:
p.e protocol (single tube):
45 min rt, 2 min inactivation; 94-60-68C for 40 cycles; 68C for 7
mins and soak at 4C
rt-pcr done with specific primers (1uM), MgSO4 1 mM
Light cycler/p.e protocol
45 min rt, 2 min inactivation (done in p.e thermal cycler) with
specific downstream primer (1uM), 5mM MgSO4
cDNA diluted 5 fold
PCR done in light cycler
MgCl2 tested - 1-4mM (levels are usually higher than that for
thermal cycler)
primers - 1uM

i would appreciate any suggestions/comments
thanks
AJ 
-----------------------------------------------------
Arul Jayaraman
Center for Engineering in Medicine
Massachussetts General Hospital/Harvard Medical School




More information about the Methods mailing list