HomeFAQsWhy is there a 55°C, 5 min heat step at the end of the assembly reaction?
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.