The primers contain uracil.
Primer Tms: 71°C (forward) and 63°C (reverse) I’ve already optimized annealing temperatures several times based on the lower Tm (63°C) but still get no visible bands. I'm using KAPA HiFi HotStart Uracil+ ReadyMix, since the primers contain uracil and are incompatible with other high-fidelity polymerases like Phusion. PCR product size: 4561 bp