qPCR – pCR2.1/RLOv Standard Curves Testing

Earlier today, I created dilution series of the following two linearized plasmids to develop qPCR assays:

  • pCR2.1/RLOv_DNA_helicase
  • pCR2.1/RLOv_head_to_tail

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).

Results:
qPCR Data File (CFX96): Sam_2015-11-06 18-17-41_CC009827.pcrd
qPCR Report (PDF): Sam_2015-11-06 18-17-41_CC009827.pdf

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.

 

Head-to-tail Curve

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.

5 thoughts on “qPCR – pCR2.1/RLOv Standard Curves Testing

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