Usage Guidelines for Golden Gate Assembly with PaqCI®

PaqCI is a Type IIS restriction enzyme that has a 7-base pair recognition sequence, useful for avoiding domestication issues in Golden Gate Assembly.  This enzyme requires more than one site to efficiently cleave.  To achieve optimal cleavage, PaqCI Activator is provided with the enzyme.  Please follow these guidelines for varying Activator requirements based on the complexity of assembly. 

See Golden Gate Assembly Protocol using PaqCI® (NEB #R0745) and NEBridge Ligase Master Mix (NEB #M1100) and Golden Gate Assembly Protocol using PaqCI (NEB #R0745) and T4 DNA Ligase (NEB #M0202) for the full protocols and more details.

Assembly with NEBridge® Ligase Master Mix (NEB #M1100)

Assembly Complexity PaqCI NEBridge Ligase Master Mix PaqCI Activator3
Single Insert Cloning or Library Prep (2 fragments)  10 U (1 µl) 5 µl 10 pmoles (0.5 µl)
Moderate assembly (3-6 fragments) 10 U (1 µl) 5 µl 10 pmoles (0.5 µl)
Complex assembly (7+ fragments) 25 U (2.5 µl) 10 µl  25 pmoles (1.25 µl)

Assembly with T4 DNA Ligase (NEB #M0202)

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.)


Other Resources

NEB Inspired: Learn how PaqCI enables 20+ fragment assembly in our blog post.