I recently optimized some newly designed primers. I used different concentrations of real-time primers (900, 450, 300, 150 nM). I got Ct values around 24-27, but the dissociation curves were jagged and irregular, and did not follow the usual one peak pattern. When I ran the products on an agarose gel, I observed only one band.
What are the possible reasons for this irregular melting curve and how can I resolve this issue?