Hi Ehsan, Use 2°C for A-T pair and 4°C for each G-C pair and calculate accordingly. I'm not sure what is your goal ? For PCR - time depends on enzyme that you use, half-life of Taq polymerase is around 30 min at 95°C, so we usually do 30 amplification cycles. For denaturation applicable time range is 30 sec to 1 min, maximum time I used for denaturation is 3 min.