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
>even greater. 
>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
-- 
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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