Master mix calcs: 20151106 – qPCR RLOv Standard Curves
All samples were run in triplicate on a CFX96 thermal cycler (BioRad).
Cycling params, plate layout, etc. can be found in the qPCR Report (see Results below).
DNA Helicase Curve
Amplifcation plots and the standard curve best fit line looks really good. Efficiency is very close to 100% and the R^2 = 0.99. Additionally, virtually all of the replicates are very tight. This looks like it will be totally usable as a standard curve for developing a qPCR assay that targets the RLOv DNA helicase gene.
This curve is way wonky. Interestingly, the end-point fluroescence levels for this curve 5-fold lower than the DNA helicase curve. I’ll likely repeat this qPCR to see if these crappy results are repeatable. However, having a single qPCR assay (the DNA helicase standard curve) for RLOv detection/quantification might be sufficient, rendering a second qPCR assay unneeded.