Looking to assemble multiple DNA fragments in a single reaction? Here are some tips to keep in mind when planning your Golden Gate Assemblies using one of our Assembly Kits for BsaI-HFv2 (#E1601) or BsmBI-v2 (#E1602), or using PaqCI®, our newest Type IIS restriction enzyme optimized for use in assembly, featuring a 7 base recognition site which minimizes the need for domestication of internal sites in your sequences.
- 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 domestication. Our tutorial video on Golden Gate Assembly Domestication provides a full description of the many options available for internal site issues. Note the use of a Type IIS restriction enzyme with a 7 base recognition site, such as PaqCI, is less likely to have internal sites present in any given 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 or assembly videos for further information regarding the placement and orientation of the sites.
- Choose the right plasmid
Consider switching to the versatile 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 best for Golden Gate Assembly with BsaI-HFv2, BsmBI-v2 and PaqCI. However, alternate buffers would be NEBuffer r1.1 for Bsa-HFv2, NEBuffer r2.1 for BsmBI-v2, or rCutSmart Buffer for PaqCI, if these buffers are 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, BsmBI-v2 and PaqCI are very stable and continue to be active during 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 for amplicon inserts/modules
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 paired with the appropriate Type IIS restriction enzyme chosen to direct your assembly to achieve high efficiencies and accurate complex assemblies. Please use the free NEBridge™ 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, refer to the NEBridge™ Ligase Fidelity Tools.
- If using precloned proven inserts that suddenly become problematic, check for a possible mutational event in your sequence
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.