I am diluting DNA samples (extracted with Macherey-Nagel NucleoSpin Tissue protocol) for qPCR analysis. I did a serial dilution by first measuring the samples with Nanodrop 2000, then based on those measurements I diluted the samples to 20 ng/µl. I measured those dilutions with Qubit 2.0 and based on those measurements diluted them to 10 ng/µl. The problem is that Qubit measured the last dilutions to have a concentration of 2 ng/µl (very consistently), instead of 10 ng/µl.

I checked my dilution calculations and I can't find a fault in them. I calculated the dilution volumes by the formula C1 * V1 = C2 * V2.

As an example, one of my samples was measured by Qubit to have a concentration of 162 ng/mL. This is 162 / 1000 * 200 = 32.4 ng/µl (I used 1 µl of sample in Qubit measurements, hence 200 / 1 ratio). To get from 32.4 ng/µl to target concentration of 10 ng/µl and target volume of 32 µl, I would calculate V1 = (C2 * V2) / C1 : (10 * 32) / 32.4 ng/µl = 9.9 ng/µl . The remaining volume (up to 32 µl) was then added as the eluation buffer used in the DNA extraction, in this case 22.1 µl .

I have done similar dilutions before with the same method, but I haven't had this problem before.

I checked the raw fluorescence measurements (RFU) for the standards and the RFU for standard 2 was 5207.05 when I was measuring the first dilutions (~20 ng/µl) and 27182.38 when I was measuring the last dilutions (~10 ng/µl). 27182.38 / 5207.05 = ~5 . Is it just a coincidence that multiplying 2 ng/µl by that ratio would give me my intended target of 10 ng/µl ? I don't see how the changing scale between standards would influence the concentration measurements, if you are measuring your samples together with the standards. Also, when I did run the samples in qPCR (to see if they would be good enough for my analysis), the amplification curves aligned close to the amplification curves of one of my qPCR standards with a concentration of ~2 ng/µl. So I doubt the Qubit measurements are wrong.

Of course there is the possibility that I did some pipeting errors when measuring concentrations and while making the dilutions, but it would be unlikely that it happened for all of my 40 samples (the concentrations measurements were quite consistent around 2 ng/µl).

Any help in pointing out some errors I have overlooked, would be appreciated before I just try doing the dilutions again.