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

NEBTV Episode 15: Restriction Enzymes in Golden Gate Assembly

Type IIS restriction enzymes have a recognition and binding site, but cut downstream of recognition site creating 4-base overhangs ideal for reassembly. Learn more at www.neb.com/TypeIISTable.

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.