Hi,
I'm in the process of developing a hydrolysis probe qPCR assay to quantify gene copy numbers of Microcystis 16S rDNA. In troubleshooting my cycling parameters, I'm getting resulting plots such as the attached photo, where a plateau phase is not reached but rather a secondary jump in fluorescence is observed in the late stages. I ran NTC samples on this run (they're labeled "Blank" in the photo), and no fluorescence was detected at all, indicating this is not due to primer dimers.
Does anyone have knowledge of certain assay parameters that may lead to a plot such as this? Possibly related to annealing or extension times/temperatures? Could inhibitors in my template cause something like this?
hi mate, this link may help you and may arise from high template concentration just dilute samples 10x - 100x and check again
https://www.researchgate.net/post/Double_plateau_amplification_plot_for_SYBR_Green_miRNA_detection
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