FAQ: When can a 37°C constant temperature assembly protocol be used instead of longer cycling protocols?

Assemblies of 1–4 precloned or PCR amplicon inserts can be performed with a 1 hr, 37°C reaction followed by 5 min at 55°C. Assemblies with larger numbers of inserts will have decreased efficiencies of assembly with this constant temperature assembly protocol compared to cycling assembly protocols that alternate between temperatures favoring BsaI or T4 DNA Ligase activity. Further variables involved in assembly are discussed in the Golden Gate Assembly Tips section.