Duplex PCR Target A & IC & target B & Ic works great. When multiplex the three together A FAM , B HEX, IC Cy5 , A & IC fine, B curve plateau strange?

The curve by B in Hex starts great but the plateau looks like zigzag. I tested different primer concentrations and different annealing temperatures. We use Lightcycler 480 II. Color compensation is on.

Any recommendations or suggestions?

Should I change the labeling?

Target A PCR is very sensitive. Target B forward primer has one mismatch but I can't change it due to logistic reasons, but it works great as a duplex with the IC (internal control)

Thanks in advance. I would appreciate any help?

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