Re: [DIYbio] Re: qPCR fluorescence detection dynamic range

> I think having even a single-tube qPCR machine coupled with some
> gelRed or gelGreen would be better than running a gel,

You'll definitely need more than one tube. You have to run controls
simultaneously to the experiments because experimental qPCR results by
themselves are meaningless and you can't directly compare data between
runs. And I would recommend doing every control and experiment in
triplicate. So if you wanted to test how a single gene changes
expression level after a certain perturbation, you would need need 3
experimental tubes and 3 control tubes before the perturbation and 3
experimental and 3 controls after the perturbation. Additionally,
unless you're working with a well established protocol like a viral
titer assay, I would recommend using multiple dilutions of your
controls.

A long time ago I was working on the ENCODE project trying to confirm
ChIP-Seq and RNA-Seq data coming out of an experimental Solexa
sequencer using qPCR. My general rule of thumb was that I needed 12
tubes to do one test if I was using a 96 well plate and 18 tubes if I
was using a 384 well plate because the smaller volume causes larger
pipetting error. So I would say the absolute minimum amount of tubes
you need for a qPCR machine would be 12.

-cory

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