Recommended Screening Protocols for NEB Golden Gate Assembly Kit (BsmBI-v2) (NEB #E1602)
- Colony PCR screening using an appropriate DNA polymerase for amplification of the insert region. Analysis primers are not included with the Golden Gate Assembly Kit. We recommend the following oligos be custom ordered through any oligo synthesis provider for colony PCR screening or sequencing of pGGAselect-based assemblies:
Forward (CW) primer 65 bp upstream from assembly point: 5′-CTGCAGGAAGGTTTAAACGCATTTAGG-3′
Reverse (CCW) primer 62 bp downstream from assembly point: 5′-TAATACGACTCACTATAGGGAGACTC-3′
Note: The suggested forward primer is identical to that recommended for assemblies using pGGA as a destination plasmid (NEB Golden Gate Assembly Kit (BsaI-HFv2) (NEB #E1601). The reverse primers differ and are specific to pGGA or pGGAselect destination constructs.
While many DNA polymerases are suitable for colony PCR, OneTaq® DNA Polymerase (NEB #M0480) or OneTaq Hot Start DNA Polymerase (NEB #M0481) is recommended. Taq DNA Polymerase (NEB #M0267) can also be used. For PCR of larger assemblies, LongAmp® Taq DNA Polymerase (NEB #M0323) or LongAmp Hot Start Taq DNA Polymerase (NEB #M0534) is strongly recommended.
- Prepare plasmid mini-preps using the Monarch Plasmid Miniprep Kit (NEB#T1010) and map by using appropriate restriction endonucleases to confirm the correct assembly.
Regardless of the screening protocol used, the correct assembly of insert(s) should always be confirmed by sequencing of the plasmid construct across the 4 base junctions and inserts. Note that larger assemblies will require internal assembly-specific primers to verify the full assembly sequence.