Any idea why are my fluorescence values not platouing at the same hight? The Cq values for the technical replicates are below 0.2 and in most cases below 0.1. Does the fact that the samples are not platouing at the same hight affect the data?
This is the first time it happened and I do not know what could be the reason I have such a scatter in the fluorescence extent.
I am studying the mitochondrial to nuclear DNA (mtDNA/nDNA) ratio using total DNA sample (undigested with RNAse A).
Nope, plateau height is entirely arbitrary. It doesn't mean much. You could run the exact same sample, on the exact same machines (if you had two of the same machine) with the same lot and batch of reagents, and you would certainly get different plateau heights. It varies from machine to machine. I wouldn't worry about it. Best of luck!
Nope, plateau height is entirely arbitrary. It doesn't mean much. You could run the exact same sample, on the exact same machines (if you had two of the same machine) with the same lot and batch of reagents, and you would certainly get different plateau heights. It varies from machine to machine. I wouldn't worry about it. Best of luck!
I agree with Craig, you should be more concerned with the Ct values of your replicates. Since PCR is a doubling process, all your replicates should have very similar Cts given that they have the same amount of starting material. This is why the blue bar is set at the exponential/linear phase, all replicates should perform or cross the threshold very similarly to each other in terms of amplification in the exponential/linear phase (assuming similar efficiency). Large deviations, however, may warrrant investigation.
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