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

Golden Gate Assembly is a one-tube efficient cloning method based on Type IIS restriction enzymes that cleave outside their recognition sites and leave 3 or 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®v2, 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. Other Type IIS enzymes used in Golden Gate Assembly include BsmBI-v2/Esp3IBbsI/BbsI-HF, PaqCI (AarI isoschizomer with 7 bp recognition sequence), and SapI/BspQI (with 7bp recognition sequence and 3bp overhangs). 

  1. KuceraBecky

    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 re-assembly. View a list of TypeIIS enzymes.