FAQ: How does NEB Golden Gate Assembly work?

Assembly utilizes two simultaneous enzymatic activities in a single reaction; endonuclease digestion by BsaI and ligation by T4 DNA Ligase. With optimized buffer components and enzyme ratios, a single reaction containing a destination plasmid such as pGGA and inserts (PCR amplicons or precloned) will result in ligation of inserts with complementary cohesive ends and the accumulation of assembled product as a function of time. The final assembly has no BsaI recognition sites, rendering the assembly inert to further digestion by BsaI. For more information, view our online tutorial at www.neb.com/goldengate.