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

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

  1. Set up assembly reactions as follows:
    REAGENT NEGATIVE CONTROL ASSEMBLY REACTION
    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:
    INSERT NUMBER SUGGESTED ASSEMBLY PROTOCOL
    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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Related Products:
PaqCI

Videos

  1. high complexity golden gate assembly targets

    Listen to DAD (Data-optimized Assembly Design) when constructing high-complexity Golden Gate Assembly targets

    This webinar introduces insights and modified protocols for Golden Gate Assembly enabling 50+ DNA fragments to be faithfully assembled in a single reaction.