Golden Gate Assembly

Why do Type IIS enzymes matter?

A conventional Type II enzyme such as EcoRI cuts inside GAATTC and always leaves the same overhang, so every fragment cut with it is compatible with every other and the assembly order is random. A Type IIS enzyme such as BsaI recognises GGTCTC and cuts one nucleotide downstream, leaving a 4 nt overhang whose sequence comes from the adjacent DNA. Different fragments therefore carry different, non-interchangeable overhangs, which is what makes ordered multi-fragment assembly possible.

How do I choose the overhangs?

Overhang choice is the single biggest determinant of whether a Golden Gate reaction works. Potapov et al. (2018) measured ligation fidelity for all 256 possible 4 nt overhangs on T4 DNA ligase and showed that some pairs mis-ligate at appreciable rates. Modern design tools score a candidate overhang set against that empirical data.

• Use overhangs that differ from each other by at least two nucleotides.
• Avoid palindromic overhangs: they ligate to themselves.
• Avoid overhang sets that are all-AT or all-GC.
• Do not reuse the same overhang twice in one reaction unless you intend those junctions to be interchangeable.
• Verify that no internal BsaI or BsmBI site exists in any fragment; domesticate the sequence first if one does.

What does the reaction look like?

Digestion and ligation are cycled in the same tube. A typical programme alternates 37 °C (enzyme cuts) and 16 °C (ligase joins) for 25–35 cycles, then a final 60 °C step to inactivate the enzyme and drive the reaction toward the assembled product. Correctly assembled junctions destroy the recognition site, so the product accumulates while unassembled fragments keep being re-cut.

Golden Gate Assembly protocol, step by step

This is the setup NEB publishes for the NEBridge Golden Gate Assembly Kit (BsaI-HFv2, E1601). The reaction is set up by molar amount rather than mass, so convert every part to pmol first.

• Destination plasmid: 0.05 pmol (75 ng of NEB's pGGAselect vector is 0.05 pmol).
• Inserts: 0.05 pmol each, a 1:1 molar ratio with the vector. PCR-product inserts need a BsaI site at each end in the correct orientation, with about 6 extra bases outside each site.
• Add 2 µl of 10X T4 DNA Ligase Buffer and 1 µl of Golden Gate Enzyme Mix (2 µl for more than 10 inserts), then nuclease-free water to 20 µl.
• 1 insert: 37 °C for 5 minutes (1 hour for library preparation), then 60 °C for 5 minutes.
• 2–10 inserts: 30 cycles of 37 °C for 1 minute and 16 °C for 1 minute, then 60 °C for 5 minutes.
• 11 or more inserts: 30 cycles of 37 °C for 5 minutes and 16 °C for 5 minutes, then 60 °C for 5 minutes. Transform competent E. coli with the finished reaction.

When should I use something else?

Golden Gate requires that no fragment contain an internal copy of the recognition site, so a sequence full of BsaI sites needs domesticating first, which costs a round of mutagenesis. If you are joining a couple of one-off PCR products and do not need a reusable part library, Gibson Assembly avoids that constraint entirely and needs no enzyme-site planning.