Golden Gate Assembly Protocol using PaqCI® (NEB #R0745) and T4 DNA Ligase (NEB #M0202)

  1. Set up assembly reactions as follows:
    Destination Plasmid1, 75 ng/μl 1 μl 1 μl
    Inserts (user provided):
    - if precloned2
    - if in amplicon form3

    75 ng each plasmid
    2:1 molar ratio (insert : vector backbone)
    T4 DNA Ligase Buffer (10X) 2 μl 2 μl
    PaqCI (R0745; 10 u/μl)4 0.5 - 2 μl 0.5 - 2 μl
    PaqCI Activator (20 μM)4 0.25 - 0.5 μl 0.25 - 0.5 μl
    T4 DNA Ligase (M0202, 400 u/μl)4 0.5 – 2 μl 0.5 – 2 μl
    Nuclease-free H2O to 20 μl to 20 μl
    1. user provided; must have 2 PaqCI sites in the proper orientation such that after cutting, the sites are not present in the vector backbone.

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

    3. Amplicon inserts must possess 5´ flanking bases and PaqCI restriction sites at both ends of the amplicon and in the proper orientation.

    4. See Usage Guidelines for Golden Gate Assembly with PaqCI for optimal amounts of PaqCI, PaqCI Activator and T4 DNA Ligase needed, based on assembly complexity.
      Note: Negative controls are not routinely done for assembly reactions, but are described for first time users.  

  2. Choose the appropriate assembly protocol based on number of inserts:
    For 1 Insert 37°C,  10 minutes (cloning) or 37°C, 1 hour (library preparation) →  60°C, 5 min
    For 2-10 Inserts (37°C, 1 minute → 16°C, 1 minute) x 30 - 60 cycles → 37°C, 5 min → 60°C, 5 min
    For 11-20+ inserts (37°C, 5 minutes → 16°C, 5 minutes) x 30 - 60 cycles → 37°C, 5 min →  60°C, 5 min
To learn more about NEB Golden Gate, please see our technical note.

You can also read about how PaqCI enables 20+ fragment assembly in our NEB Inspired blog post

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