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  • 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 4-base overhangs.  (Ref 1:  Engler C, Kandzia R, Marillonnet S (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3(11): e3647. doi:10.1371/journal.pone.0003647. PMID:18985154) BsaI is one Type IIS enzyme used in this method.  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.  BsaI-HF works in CutSmart™ Buffer, as does T4 DNA Ligase (also part of the Golden Gate Assembly workflow) which is 100% functionally active  in this buffer. Other Type IIS enzymes used in Golden Gate include BspMI and BfuAI.