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 table below, or Usage Guidelines for Golden Gate Assembly with PaqCI, for optimal amounts of PaqCI, PaqCI Activator and T4 DNA Ligase needed, based on assembly complexity.   
      
      

Assembly Complexity	PaqCI	T4 DNA Ligase	PaqCI Activator3
Single Insert Cloning (10 min 37°C) or Library Prep (60 min 37°C)	5 U	200 U	5 pmoles  (1/4 µl of a 20 µM stock)
Simple to Moderate assembly1 (2-10 fragments)	5-10 U	200-400 U	5 pmoles   (1/4 µl of a 20 µM stock)
Complex assembly2 (11-20+ fragments)	10-20 U	400-800 U	5-10 pmoles   (1/4-1/2 µl 20 µM stock)

 

1. Based on 5 fragment assembly test system results
2. Based on 24 fragment assembly test system results
3. The activator solution is in a Mg-free buffer for optimal long-term storage. For short-term working stocks, if desired, dilute an appropriate amount in 1X T4 DNA Ligase Buffer to achieve more easily pipettable volumes (e.g., a four-fold dilution = 5 µM , 5 pmoles/µl activator.)
    
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

For higher efficiency and accuracy, please see Golden Gate Assembly Protocol using PaqCI® (NEB #R0745) and NEBridge Ligase Master Mix (NEB #M1100)

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