Initial step:	3 min	95°C	(denaturation)
25 cycles:	30 s	95°C	(denaturation)
	30 s	50-65°C*	(annealing)
	1 min/kb**	72°C**	(extension)
Final step:	5 min	72°C	(extension).

^*The annealing temperature will depend on the melting temperature of the primers (see, e.g., https://www.sigmaaldrich.com/technical-documents/articles/biology/oligosmelting-temp.html). For best results, set up multiple PCRs at different melting temperatures and choose one that provides a single clean band on the gel.

^**If the plasmid is >10 kb, increase extension time to 2 min/kb and reduce temperature to 68°C.