How to measure Cy5-primer concentration ??
ivanoov at gmail.com
Fri Oct 6 05:15:05 EST 2006
Wow, what a discussion happened here ! So, for a common multiplex PCR
with 6 primer pairs (that is 6 5'Cy5 primers) I assume there is no need
for extreme accurate quantitation of Cy5 absorption at 646 nm. Moreover
and unfortunately, our spectrophotometer range is up to 600nm.
Thanks all of you
Peter Ellis wrote:
> DK wrote:
> > <pjie2 at cam.ac.uk> wrote:
> >> DK wrote:
> >>> Cy5 almost certainly absorbs srtongly at 260-280 (two indole rings).
> >> It doesn't.
> > Sure it does. What is there in Cy5 structure that indoles suddenly
> > would
> > stop absorbing ultraviolet? Nothing. Sorry, I don't believe it.
> Go and try it then. In the context of fluorescently labelled DNA, Cy5
> fluorescence at ~260 is negligible. Every group working with labelled DNA
> knows this (or should). It's how you quantitate the efficiency of your
> labelling - you measure the DNA concentration by looking at 260, the Cy5 (or
> Cy3) concentration by looking at their absorption maxima, etc.
> The signals do not interfere.
> >WRT the original question, quantifying based on Cy5 absorption is
> >definitely more precise approach.
> Negligibly more precise.
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