Hello, I am trying to confirm ligation (after cloning) via colony PCR using GoTaq Green Master Mix. Tm of FP and RP is 50C and 58C, respectively. Applied annealing temp so far 44C, 48C, 50C, 52C, 55C, 58C, 60C, 63C. There are no colonies on negative control. Any suggestions to improve?

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