Currently, I am working on my thesis, which is about the development of primer-specific species for turtles using the ND2 gene mtDNA as a gene target. I was trying to make a standard curve using a blood sample as a positive sample and used a ten-fold serial dilution. Unfortunately, the Cq for the first dilution is high. Could someone enlighten me on why it is possible? Is it possible due to a lack of a copy number gene target or an inappropriate design primer?

I’ve tried to increase the annealing temperature up to 53˚C, but the result is getting worse regarding the Cq value and graph of the standard curve.