Gibson Assembly™ employs three enzymatic activities in a single-tube reaction: 5´ exonuclease, the 3´ extension activity of a DNA polymerase and DNA ligase activity. The 5´ exonuclease activity chews back the 5´ end sequences and exposes the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed regions. A DNA ligase then seals the nick and covalently links the DNA fragments together. The overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies. The NEB Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Cloning Kit (NEB #E5510S) enable rapid assembly at 50˚C.
Tags DNA Modifying Enzymes, Cloning & Synthetic Biology, Usage Guideline