We are trying to adjust a ddPCR in duplex with two probe set: FAM and VIC. The 2D plot demonstrate 4 droplet fractions but all seems to be detected in channel 1 (FAM) even the VIC+ droplets. We changed many things in reaction but the result is always the same. We even tried to use another QX200 machine but again the same result (in-attached).
Do any one have any idea about the reason,
Dear Shabi,
If it is a rare mutation detection study, maybe it's about the probes themselves.
Did you try to run a ddPCR in the samples that only contain wild-type?
And did you try at different temperature? Normally I would run a gradient, so I'll know the best Tm.
Also it seems like you manually analyze droplets. The amplitude of the grey empty droplets is -2000. Normally it is above 0.
Good luck!
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