Golden Gate Assembly Protocol for using NEB Golden Gate Assembly Kit (BsmBI-v2) (NEB #E1602)

  1. Set up assembly reactions as follows:

    REAGENT ASSEMBLY REACTION
    pGGAselect Destination Plasmid (1), 75 ng/μl 1 μl
    Inserts (user provided):
    - if precloned (2)
    - if in amplicon form (3)
     
    75 ng each plasmid
    2:1 molar ratio (4)
    (insert:vector backbone; pGGAselect = 2,155 bp; 75 ng = 0.05 pmol)
    T4 DNA Ligase Buffer (10X) 2 μl
    NEB Golden Gate Enzyme Mix (BsmBI-v2) 1–2 μl (5)
    Nuclease-free H2O to 20 μl (6)  

    (1)  or user provided.

    (2)  Precloned inserts must possess BsmBI restriction sites at both ends of the insert sequence and in the proper orientation.

    (3)  Amplicon inserts must possess 5 ́ flanking bases (6 recommended) and BsmBI restriction sites at both ends of the amplicon and in the proper orientation.

    (4)  The NEBiocalculator® Tool (nebiocalculator.neb.com) can be used for molar calculations.

    (5)  For assemblies ≤ 10 inserts, use 1 μl ; for assemblies > 10 inserts, use 2 μl.

    (6)  Can be increased to 25 μl volume if required due to DNA component volumes; add additional 0.5 μl T4 DNA Ligase Buffer (10X)

  2. Choose the appropriate assembly protocol:

    INSERT NUMBER SUGGESTED ASSEMBLY PROTOCOL
    For 1 insert 42°C, 5 min (cloning) or 42°C, 1 hr (library preparation) → 60°C, 5 min
    For 2–10 inserts (42°C, 1 min → 16°C,1 min) x 30 → 60°C, 5 min  
    For 11–20+ Inserts (42°C, 5 min → 16°C, 5 min) x 30 → 60°C, 5 min