FAQ: Why is there a 55°C, 5 min heat step at the end of the assembly reaction?

The final incubation step at 55°C favors BsaI cutting, in the absence of DNA ligation. Digesting any plasmid still present in the assembly reactions reduces background. Golden Gate Assembly can tolerate one internal BsaI site in one fragment; in this case omit the 55°C step and screen more colonies as the background will be higher.