Editing QPCR mix (section)

=== Using the Pfaffl Method ===

*This is the prefered method as it corrects for differences in PCR efficiency as well as amplicon length without calibration.
*You should probably run a dilution just to see if your values jive.
*This method could be used to measure differential gene expression as well as comparative populations.

1. Definitions:

::
*Target - The population you're trying to quantify
::*Reference - The population you're comparing it to
::*Control - A treatment for which you are measuring both target and reference populations
::*Sample - A treatment for which you are measuring both target and reference populations comparing to the control treatment

2. Define an efficiency for each reaction using the slope during the exponential phase of the reaction:

::
*Efficiency = E = 10<sup>-1/slope</sup>.
::*This is the major difference with the previous method, and corrects for differences in amplicon length as well as copy number.
::*The slope of different dilutions (if you made any) should be the same.

3. Define the ΔCt for target and reference populations (the Pfaffl paper uses "CP" instead of Ct).

::
*ΔCt<sub>target</sub> = Control Ct<sub>target</sub> - Sample Ct<sub>target</sub>.
::*ΔCt<sub>reference</sub> = Control Ct<sub>reference</sub> - Sample Ct<sub>reference</sub>.

4. Compare the sample to the control using the ΔCt's

::
*Ratio = (E<sub>target</sub>)<sup>ΔCt<sub>target</sub></sup> / (E<sub>reference</sub>)<sup>ΔCt<sub>reference</sub></sup>
::*This is the ratio of the target in the sample to the target in the control, using the reaction effiency (E) and the reference levels to normalize.

http://www.top-bio.com/qpcr-master-mixes-48.html?qpcr-2x-master-mix-1405#breadCrumbs
The Mix is 2x concentrated: 20 mM Tris-HCl, pH 8. 8 (at 25oC), 100 mM KCl, 0.2 Triton X-100, 3 mM MgCl2, 400 µM dATP, 400 µM dCTP, 400 µM dGTP, 400 µM dTTP, Taq DNA polymerase (50 U/ml), monoclonal antibody anti-Taq, stabilizers and additives.

 stabilizers and additives.