Hi,

Do you have the simplex image of the clouds too, with the same primer concentrations? (I also cannot read the concentrations due to the resolution)

I think that these might be options for an explanation:

- either your concentration is too low to detect double positives --> if you put more DNA, an extra population will pop up (sorry, I cannot read the concentrations)

- there might be populations falling together. If you play around with the primer concentrations, they should separate out

Caroline Weydert

Hi, thank you very much for your answer. I am attaching the files, hopefully in better resolution. The input concentration was 50ng gDNA from fresh tissue isolation and other duplexes showed good separation of clusters with the same amount of DNA. The target amplicon is 107bp and the reference is 73. Could this possibly be an issue due to preference amplification?

Hi,

Thanks! Numbers look pretty similar with the singleplex and duplex.

I think that it might be a matter of concentration. If you add more DNA, do the double positives appear? (Or do you see a linear trend if you make a dilution series?)

KR,

Caroline