I am running RT-qPCR for a gene. However I still see amplification with lower Cq values in my negative control. Previously the Cq values of the negative controls were above 30. But now it averages at 24. I have diluted my primer concentration below the SYBR Green Supermix primer concentration range (250-500nM) but I still see amplification in my negative control. Initially I had thought there might be contamination of my water or primers and reagents and so I got new primers, reagents and water but I am still getting the same results from the negative control. What could account for the change in Cq values of my negative control? Does anyone have experience running RT-qPCR for B1 gene of Toxoplasma gondii?
Thank you
You have contamination from previous amplified material getting into your assay, You will need to clean your working area and pipettes and change all reagents and water and set up the assay in a new area with different pipettes
If the Cqs were around 30 before and are 24 now, that could mean there's more copies of whatever is being amplified compared to before which would make sense if someone is amplifying materials (via PCR). I've seen aerosolized contaminants from PCR amplicons show up in NTCs before.
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