Episode 55: Molecular Cloning Series: Golden Gate Assembly 101

Andy explains that Golden Gate assembly is a restriction enzyme-based cloning method using type 2S enzymes to assemble multiple DNA fragments in a predetermined order in a single reaction, unlike traditional type 2P restriction enzymes.

Andy describes the practical uses of Golden Gate assembly in synthetic biology for mixing molecular components like promoters, genes, and terminators, and highlights its advantages including low empty vector backgrounds and ability to assemble up to 30 parts reliably.

Andy outlines different ways to perform Golden Gate assembly, including standardized hierarchical assembly methods organized in multiple rounds and combinatorial assembly for creating variant libraries, emphasizing the importance of developing screening strategies.

Andy explains the need to domesticate sequences by removing native type 2S restriction sites from DNA, and introduces PacI1 enzyme with a seven base recognition site to reduce domestication needs.

Andy describes available NEB tools including the anyBridge Golden Gate visualization tool for checking assemblies, the Ligase Fidelity tool for diagnosing problems, and the anyBridge Ligase Master Mix kit that simplifies the assembly process.