Table 2. Summary of optimal TPA conditions.

Factor	Unit	Tested conditions	Optimal condition
Junction TM	°C	35, 40, 45, 50, 55, 60, 65	50
Annealing temperature	°C	T M - 10, TM - 5, TM, TM + 5, TM + 10	T M + 5
Protocol		one-step, two-step
DNA inputs	fmol/μl/frag	0.5, 1, 2, 4	2
DNA ratios	insert:backbone	1:1, 1:2	1:1
Incubation time	min	30, 60, 90, 120	120