FAQ: What affects the efficiency of Golden Gate Assembly?

Single insert cloning is more efficient than multiple insert cloning. Assembly efficiency decreases as the number of fragments increases. The presence of repetitive sequences in an insert will also decrease efficiency. For inserts < 250 bp or > 3 kb, precloning will increase efficiency. Lastly, the normal restrictions on overall plasmid size to allow stable maintenance in E. coli apply to Golden Gate Assemblies. Efficiencies are highest with assembled product plasmid constructs < 12 kb. Larger assemblies can be made but will require larger numbers of colonies to be screened for the correct full length assembled products. For experimental examples of complexity vs. efficiency, refer to the Golden Gate Assembly Technical Note.
KuceraBecky

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.