Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Qubit 2-point Standard Curve- Hill Plot - (Sep/03/2013 )

The Qubit 2.0 Fluorometer High-Sensitivity kit for DNA quantification relies on a 2-point standard curve to generate your data.


The appendix indicates the Qubit uses a "modified Hill Plot" using your 2-point standard curve to generate the regression equation to backfit your sample data.


I found a paper on making Hill Plots but it's way too math-heavy for me to follow.


I've used the kit components to make a standard curve with various points between the 2 already provided with the kit and have observed a non-linear relationship between the DNA input and the Green RFU values. Obviously, a simple linear regression is not appropriate for backfitting the data, and yet I don't understand how the fluorometer can be doing anything else since it only uses a 2-point curve.


My question, how can you generate a regression equation that is nonlinear using only 2 points on a standard curve?



-David Gray Lassiter-

Basically, as I understand it (similar to you) you can't, but you could use a pre-designed/idealised curve that takes into account two (critical?) points and uses those to extrapolate the rest of the curve, especially in the case where you know the kinetics of the reaction - as Invitrogen surely do for the Qubit.