Why am I seeing this ROX drop during a SYBR melt?

Go to the profile of alexmcfadden
Feb 09, 2018
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Hello! First post here.

I am having trouble with high levels of early noise in my SYBR Fast experiments. I notice a drop in ROX and corresponding rise in my base signal during the dissociation phase, which makes no sense to me.
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Specs:
5uL sample (low EDTA TE as the elution buffer), 5uL SYBR Fast, 0.02uL forward primer, 0.02uL reverse primer in each well
7900HT machine, 384-well plate
ROX in SYBR Fast is 500nM final concentration

Sometimes I see this pattern, and other times it's very clean and starts flat like it should. Any advice?

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