I have been having no success in getting the expected band size. My expected product is 2272 bp in length. I did fusion PCR in the first stage with no primers for 15 cycles (annealing temp 60), then used 5ul of the product for the next stage, include a set of primers, for 30 cycles. I used a high fidelity mastermix for both stages. I tried 3 annealing temperatures at this point (58, 60, 65). When I ran the gel, the band appeared at 1500bp (temp 58) while the rest showed no bands. Please suggest on how I can improve. Thank you.