Hi, usually master mix producers indicate a generic protocol for concentrations (i.e. range 50 ng -5 pg of gDNA and 250-500nM range for primers)  and temperatures. For the initial optimization, you should perform serial-diluition of your template and evaluate the curves. As well, you should perform a annealing temperature gradient that allow you to evaluate several annealing T in a single experiment. Good luck

You need to first calculate the Tm of your primer sequence (Base stacking Tm preferably). Once you know the Tm, run a gradient PCR with annealing temperature near your calculated Tm (above and below the calculated value). Run the product on a gel against a marker and select the temperature with least or no non-specific products. Similarly, you can try different concentrations of DNA for getting a band of proper intensity.

Good luck!  

Thanks a lot to all of you for your help

I already tried a gradient PCR  around the annealing temperature I calculated and no amplified product appeared on the gel!!

I think the product length of the primers is too short to appear without a probe so I will design another reverse to make the product length around 150 bp

The other option is to try another gradient PCR with a higher range of temperatures!

Hi,

The optimum product size is better to be between 75 to 200, in an intercalating dye strategy. A 52 bp product size is very close to the size of probable primer dimmers in amplification runs and might cause other problems during the setup process as well as melting cure analyses. 

Good luck with the new set!

You are right Riham El Sayed, try primers which can give 120-150 bp products. It is best suited for RT-PCR work.