Hi all,
May I have some suggested reasons why I am obtaining amplification curves (in pink) with unusual shapes (particularly near the baseline) and plateaus of various heights?
This is a Taqman-based duplex assay. One probe contains VIC-TAMRA, while the other contains FAM-TAMRA. Only one of the targets is present in the PCR.
Thank you.
Hi Nathanael,
Ur qpcr curves seem fine. As i see from your attachments, u have not set any threshold baseline.. set a threshold either automatically or manually. So that it lies at level of 0.4-0.5 of delta Rn because at that particular level, ur products have started to form (24-25cycles in X axis). Regarding, different heights of the three replicates in ur amplification curve, its simply because of manual error in pipetting that has caused formation of different amount of products in each well of triplicate. And so u get peaks at differnet level
Hi Subhasri Biswas , thanks for your answer. However, doesn't it seem strange that some curves do not follow a rectangular hyperbolic shape closely? I was wondering if (1) this is normal (2) what are the possible causes of these shapes.
This is another set of triplicates that I obtained recently. One of the curves does not follow a rectangular hyperbolic shape like the others.
It looks pretty normal(red curves), which is your the value for the baseline, and the values for the Cts (apply a threshold).
How much template did you put in your reaction?
This just looks like noise, combined with a rather high level of background fluorescence to me. It's worth remembering that logarithmic plots tend to amplify small variations around near-zero fluorescence. I suspect that if you did a linear plot, you would see nothing anomalous - though I prefer log plots for precisely this reason.
And a Happy New Year to you all!
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