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. These need to have any internal sites eliminated by site directed mutagenesis; the Q5® Site-Directed Mutagenesis Kit, (NEB #E0554S) and the NEB web tool NEBaseChanger® work well for this purpose.
- 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 manual for further information regarding the placement and orientation of the sites.
- Choose the right plasmid
Consider switching to the pGGA Destination Plasmid for your Golden Gate assembly. The pGGA plasmid is included in the Golden Gate Assembly Kit (NEB #E1601). It has T7 and SP6 promoter sequences flanking the assembly site, and has no internal BsaI sites. The pGGA 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 BsaI-HF® v2, however NEBuffer 1.1 supplemented with 10 mM DTT and 1 mM ATP also supports assembly.
- Increase your complex assembly efficiency by increasing the Golden Gate cycling levels
T4 DNA Ligase and BsaI-HFv2 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 45-60, 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 over-estimation 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 efficiency of assembly.
- Increase enzyme amount for complex assemblies
For complex assemblies involving >10 fragments, increasing the BsaI-HFv2 and T4 DNA ligase levels two-fold will enhance the efficiency of assembly.
- Carefully design EVERY insert’s overhang
Regardless of complexity, assembly efficiency is only as good as its “weakest link”. Make sure every pre-cloned insert/module or amplicon has well-designed overhangs that are unique, nonpalindromic, and not predicted to form significant mismatch ligation products with any other overhangs in your assembly (https://pubs.acs.org/doi/10.1021/acssynbio.8b00333). Use Ligase Fidelity Viewer to visualize overhang ligation preferences for T4 DNA ligase under various experimental conditions.
- 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.