I am doing qPCR over and over again exactly based on its protocols and I sure about quality of primers but every time that I am doing qPCR I cant not get sharp pick some times I have two picks and some time it is wide is there any body help me?
Why are you sure about the quality of the primers when this data is telling you that the primers are likely bad? Have you run the product on a gel to see if a clean band is obtained? You could try running gradient PCRs to optimise the annealing temperature to see if it's possible to clean up this primer set but it may be necessary to redesign the primers. Do you have any other primer sets that are giving you the expected results during the melting analysis?