FAQ: Which restriction enzymes are used in Golden Gate Assembly?

Restriction Enzymes in Golden Gate Assembly

Type IIS restriction enzymes have both recognition and binding sites, but cut downstream of the recognition site, creating 4-base overhangs ideal for reassembly. View a list of TypeIIS enzymes.

Golden Gate Assembly is a one-tube efficient cloning method based on Type IIS restriction enzymes that cleave outside their recognition sites and leave 4-base overhangs. BsaI is the most commonly used Type IIS enzyme for Golden Gate Assembly. NEB also offers an engineered high-fidelity version of this enzyme, BsaI-HF, which has the added benefit of being Time-Saver™ qualified (can digest DNA in 5-15 mins or overnight without degradation to DNA) and exhibits dramatically reduced star activity; however methylation sensitivities differ between the two enzymes. Presently BsaI, rather than BsaI-HF, is more commonly used in Golden Gate published protocols. Other Type IIS enzymes used in Golden Gate Assembly include BsmBI and BbsI.