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

Reading a CCD datasheet just now, it defines dynamic range (DR) as
DR=Vsat/Vmdk... where Vsat is the saturation output voltage, and Vmdk
is the maximum dark noise voltage

On Thu, May 23, 2013 at 3:43 PM, Cory Tobin <cory.tobin@gmail.com> wrote:
>> 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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--
-Nathan

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