perkin elmer vs light cycler
Joan Marie Shields
jshields at taurus.oac.uci.edu
Wed Aug 25 11:47:52 EST 1999
Weining, Song <WeininS at prose.dpi.qld.gov.au> wrote:
>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
>It will take too long to list all the details here and I have been working
>on a book to discuss it.
Tell me about it. I've been trying to get the Idaho Light Cycler we
have to work consistantly. We use the microtubes rather than the
capillary - not only is it easier but we're woking with environmental
samples so the capillary tubes just won't work with what we need (even
the 50microL ones).
So far I've found that the annealing temperature and the MgCl2
concentrations are tied together. Go up a degree and you have to
increase the concentrations by at least a 0.5mM. I'd prefer to keep
the MgCl2 down if I can.
I've also noticed that my yield is lower which makes the detection
limit much higher than for the block machines.
What I'd like to know is given the shortened time to ramp give the
sample inside the tube enough time to equilibrate? I'm holding the
samples for 20 seconds for both denaturation and annealing (during
cycling) which is a mere 10 seconds less than I would use for the
block machine. I've also cut the extension time down from 1 minute
to 30 seconds (1 second per 25 nucleotides + 4 seconds = 650bp amplicon).
Is this enough?
Also, is there a newsgroup devoted to this?
Joan Shields jshields at uci.edu http://www.ags.uci.edu/~jshields
University of California - Irvine School of Social Ecology
Department of Environmental Analysis and Design
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