Looking to assemble multiple DNA fragments in a single reaction? Here are some tips to keep in mind when planning your Golden Gate Assembly experiment.
- Check your sequences
Always check your assembly sequences for internal sites before choosing which Type IIS restriction endonuclease to use for your assembly. While single insert Golden Gate assembly has such high efficiencies of assembly that the desired product is obtainable regardless of the presence of an internal site, this is not true for assemblies with multiple inserts. Options include choosing a different Type IIS restriction enzyme to direct your assembly, or eliminating internal sites through mutagenesis. The Q5® Site-Directed Mutagenesis Kit (NEB #E0554S) and the NEB web tool NEBaseChanger work well for this purpose. Alternately, a junction point can be created at the internal site’s recognition sequence.
- Orient your primers
When designing PCR primers to introduce Type IIS restriction enzyme sites, either for amplicon insert assembly or as an intermediate for pre-cloning the insert, remember that the recognition sites should always face inwards towards your DNA to be assembled. Consult the Golden Gate Assembly Kit manuals for further information regarding the placement and orientation of the sites.
- Choose the right plasmid
Consider switching to the pGGAselect Destination Plasmid for your Golden Gate assembly. This versatile new destination construct is included in all Golden Gate Assembly kits and can be used for BsaI-HFv2, BsmBI-v2 or BbsI directed assemblies. It also features T7 and SP6 promoter sequences flanking the assembly site, and has no internal BsaI, BsmBI or BbsI sites. The pGGAselect plasmid can also be transformed into any E. coli strain compatible with pUC19 for producing your own plasmid preparation if so desired.
- Choose the right buffer
T4 DNA Ligase Buffer works well for Golden Gate Assembly with both BsaI-HFv2 and BsmBI-v2. However, alternate buffers would be NEBuffer 1.1 for Bsa-HFv2 and NEBuffer 2.1 for BsmBI-v2, as long as supplemented with 1 mM ATP and 5-10 mM DTT.
- Increase your complex assembly efficiency by increasing the Golden Gate cycling levels
T4 DNA Ligase, BsaI-HFv2 and BsmBI-v2 are very stable and survive extended cycling protocols; an easy way to increase assembly efficiencies without sacrificing fidelity is to increase the total cycles from 30 to 45-65, even when using long (5-minute) segments for the temperature steps.
- Make sure your plasmid prep is RNA-free
For pre-cloned inserts/modules, make sure your plasmid prep is free of RNA to avoid an overestimation of your plasmid concentrations.
- Avoid primer dimers
For amplicon inserts/modules, make sure your PCR amplicon is a specific product and contains no primer dimers. Primer dimers, with Type IIS restriction endonuclease sites (introduced in the primers used for the PCR reactions), would be active in the assembly reaction and result in mis-assemblies.
- Avoid PCR-induced errors
Do not over-cycle and use a proofreading high fidelity DNA polymerase, such as Q5® DNA High-Fidelity Polymerase.
- Decrease insert amount for complex assemblies
For complex assemblies involving >10 fragments, pre-cloned insert/modules levels can be decreased from 75 to 50 ng each without significantly decreasing the efficiencies of assembly.
- Carefully design EVERY insert’s overhang
An assembly is only as good as its weakest junction. Research at NEB has led to an increased understanding of ligase fidelity, including the development of a comprehensive method for profiling end-joining ligation fidelity in order to predict which overhangs will result in improved accuracy. This ligase fidelity information can be used in conjunction with the NEB Golden Gate Assembly Kits (BsaI-HFv2 or BsmBI-v2) to achieve high efficiencies and accurate complex assemblies. Please use the free NEB Golden Gate Assembly Tool to design primers for your Golden Gate Assembly reactions, predict overhang fidelity or find optimal Golden Gate junctions for long sequences. When working with complex assemblies ( >20 fragments), refer to the ligase fidelity tools on the NEBeta Tools site.
- Check for a sequence error if your assembly becomes non-functional
Be aware that occasionally a pre-cloned insert/ module can become corrupted by an error during propagation in E. coli, usually a frameshift due to slippage in a run of a single base (e.g., AAAA) by the E. coli DNA Polymerase. This should be suspected if previously functional assembly components suddenly become nonfunctional.