Yeah, I also recommend the answer provided by, Abhijeet Singh . Dear Subash Thapa The range of annealing temperatures of the primer pair which you have selected has been recorded in the user manual. find the range and do your amplification starting the lowest temperature and gradually increase the temperature at the annealing step. within this range you can find the best annealing temperature giving the best amplified product. You can refer the most recent articles relevant to the both primer pair and the species to find the best annealing temperature. If you have further difficulties Trouble shooting will also help you to find possible clarifications.