Hello everyone,

I am unfortunately getting sub-quality results from my dPCR runs where the clustering (i.e. fluorescent amplitude) of the FAM and VIC assays are not separated. Essentially, I am getting the FAM, VIC, FAM+VIC, and no reaction clusters all forming in the same area instead of them forming distinct clusters as expected (please see attached image).

In another run, I had only clusters corresponding to FAM and FAM+VIC.

I am using the QuantStudio 3D workflow for rare allele detection. The protocol recommends a working concentration of 200-2000 copies/uL of DNA in the final reaction.

Could this issue be associated with insufficient DNA concentration in the final reaction, I am currently using 1ng/uL (I am not quite sure how to relate this to the copies/uL units above)? Or could this be due to oiling the chip incorrectly?

Any insight provided by anyone with experience in dPCR would be greatly appreciated! It seems that the rare allele is indeed present (due to FAM clusters), but the clusters are just not forming correctly.

Thank you in advance.