Re: [DIYbio] Re: Phosphorescent PCR dye

On Wed, May 6, 2015 at 10:30 AM, Gabriele <blueslevin@gmail.com> wrote:
> Thank you for your answer and for the documents.
> I think that phosphorescent could be useful because it is slow. It could be
> possible to excite the solution, turn off the led/laser and then measure the
> emission. This will avoid the use of optical filters, which are quite
> expensive. It's possible to find high speed photodiode which they can
> measure Fluorescence too, but thery are expensive and not so accurate. So I

Are optical filters really expensive? I know that in previous
generations of qPCR machines, they used lasers which needed 220V. The
newer LED machines can use regular 110V so I think that makes it less
expensive. I think a replacement laser for the Applied Biosystems
7900HT costs between $10000 to $15000.

> think phosphorescence cuold be useful in this application. How longer does
> the phosphorescence take from the excitation to the emission? And for how
> longer does it emit?
>

Since the amplification part of qPCR (i.e. PCR) doesn't take too long,
this might where you want to look. Amplification has denaturation,
annealing, and extension. In most cases, you detect fluorescence at
the end of extension. The per cycle time is probably around a minute.
The Applied Bio Quantstudio already has the predefined optimized
cycling program that works with their dyes and that goes under 30 sec.
I worked with Roche Lightcycler before where you had to optimize the
PCR conditions. My notes for those experiments say they cycle for
under 30 sec.

The Applied Bio QuantStudio actually detects the signal thru all 5
filters it has (even though you may only have one type of probe on the
whole plate). I was told this whole process (all 5 filters) occurs in
nanosecond (or pico?). It's very fast.

So if you want to use phosphorescence, you might have to figure out
how to quench the emission signal before the next collection point.

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