real time PCR

Dr. Duncan Clark news at
Thu Jul 5 14:21:39 EST 2001

In article <3B44B233.9F9EE260 at>, the eminent Rui Pires
Martins at Center for Molecular Medicine and Genetics @ WSU School of
Medicine wrote
>My question is then, when (quantitatively) declaring a fragment more
>prevalent in one fraction over another, is the critical parameter the
>maximum fluorescence of the sample, or should the cycle at which the
>sample starts to amplify also be considered?

Ct (or the cycle at which you just start to see amplification) is the
critical value. That is the stage at which are you still seeing
exponential amplification and nothing is yet rate limiting i.e. the Ct
value is copy no. dependant. At the plateau phase all you are really
measuring is the difference between how well one PCR panned out compared
to the next identical PCR. If you run say 96 identical PCRs from one
complete master mix you will find that the Ct for all 96 are virtually
identical whilst the final fluorescence varies enormously.

There is a 7700 listserv and a LightCycler listserv where you can ask
the really experienced labs. I am subscribed to both but am pushed for
time now to re-hunt up the listserv addresess. A quick search using say
Google will find them both.

Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR: 

Optimisation, Probe Technology & Future Systems

4-5 September 2001
King Alfred's College, Winchester, UK

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