Historically, Golden Gate inserts were precloned into plasmid constructs having flanking Type IIS restriction sites to generate the appropriate 4 base overhang sequences that guide the assembly. However, the use of amplicon inserts without precloning also supports efficient assembly levels and saves time. See below for specific recommendations for precloned inserts, and amplicon inserts for single insert cloning and multiple insert assembly:
A. Precloned Inserts: Precloning is always an option, and is superior for inserts < 250 bp or > 3 kb, or those containing repetitive elements that might accumulate errors during PCR amplification. Note that all sequences that will be part of the assembly must be flanked by correctly oriented BsmBI restriction sites, facing towards the insert on the top and bottom strands.
B. Amplicon Inserts: The 5′ flanking bases and BsmBI restriction enzyme recognition site are introduced through PCR primer design upstream and downstream of sequences to be assembled. In all cases, the 2:1 insert:vector backbone (pGGAselect, 2,155 bp) molar ratio is suggested to achieve assembly efficiencies similar to that with precloned inserts.
(a) Single Insert Cloning/Assembly Primer Design:
Single insert amplicons should be single specific PCR products, with no non-specific amplification or smearing present; if the amplicon is not present as a single product, optimize the PCR amplification.
While single insert amplicons can be used directly from PCR without purification under certain circumstances (see FAQ), it is always best to purify amplicons using spin columns such as the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030). Use of these kits will result in purified, higher concentration DNA due to smaller elution volumes.
(b) Multiple Insert Assembly Primer Design:
The first and last inserts will require the same four base overhangs diagrammed above for single insert assembly as these overhangs will ligate to the pGGAselect vector backbone. For all other inserts involved in the multiple insert assembly, we recommend using the NEB Golden Gate Assembly Tool (goldengate.neb.com) for the design of PCR primers to ensure the correct unique 4 base overhangs between inserts.
All amplicons should be single specific PCR products, with no non-specific amplification smearing present; if the amplicon is not present as a single product, optimize the PCR amplification.
All multiple inserts must be purified using spin columns as described above.