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

Go to the profile of alexmcfadden
Feb 09, 2018

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.
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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