The melting temperature and primer annealing temperatures are important because the higher the annealing temperature in a pcr the more specific the annealing and, therefore, the pcr product becomes. Annealing at too low a temperature leads to many non-specific bands and too high an annealing temperaure leads to no product at all. The melting temperature is an approximate guide to what temperature to run the annealing step of the pcr so at Tm minus 5 degrees C you can expect to generate a pcr product and at temperatures higher than this you should get a cleaner amplification up to a temperature when the pcr fails because the primer cannot anneal

Based on my experiment, I ran a set of PCR at 60℃ and observed that the number of targets continued to decrease as the primer Tm (melting temperature) was reduced. This suggests that the primers were not able to bind effectively to the target DNA sequence, resulting in a lower yield of PCR products. The naming convention "group*_L_50" indicates that the primer set belongs to a specific group and has a Tm (melting temperature) of less than 50℃ and greater than 50-5℃ as determined by Primer3 software. It is important to note that if the Tm of the primers is less than 45℃, there is no PCR products generated at an annealing temperature of 60℃.