Golden Gate Assembly Protocol using PaqCI^® (NEB #R0745) and NEBridge Ligase Master Mix (NEB #M1100)

1. Based on assembly complexity, set up assembly reactions as follows:
   

   COMPONENTS	2 FRAGMENT OR 3–6 FRAGMENT ASSEMBLY	7+ FRAGMENT
   NEBridge Ligase Master Mix (NEB #M1100)	5 μl	10 μl
   DNA Fragments*	0.05 pmol each	0.05 pmol each
   PaqCI (NEB #R0745)	1 µl (10 U)	2.5 µl (25 U)
   PaqCI activator (20 µM)	0.5 μl	1.25 μl
   Nuclease-free Water	x μl	x μl
   Total Reaction Volume	15 μl	30 μl

   * Use NEBiocalculator^® to calculate the mass of each DNA fragment

2. Set up a reaction in a microcentrifuge tube on ice. Mix DNA fragments (0.05 pmol of each) with nuclease-free water (x µl).
3. Add NEBridge Ligase Master Mix (5 µl or 10 µl) to DNA fragments and water. Gently mix by pipetting 3 times. 
4. Add PaqCI activator (0.5 µl or 1.25 µl) then PaqCI (1 µl or 2.5 µl) to the reaction. Gently mix by pipetting 5 times. 
5. Incubate for the recommended time and temperature:
   

   2 FRAGMENT ASSEMBLY		3–6 FRAGMENT ASSEMBLY	7+ FRAGMENT ASSEMBLY
   SINGLE GENE CLONING	LIBRARY CONSTRUCTION		7–13 FRAGMENT	14+ FRAGMENT
   37ºC for 15 min.	37ºC for 60 min.	30 cycles at 37ºC for 1 min. and 16ºC for 1 min.	30 cycles at 37ºC for 5 min. and 16ºC for 5 min.	60 cycles at 37ºC for 5 min. and 16ºC for 5 min.



6. End Soak: Incubate at 60°C for 5 minutes, before transformation.
7. Chill on ice.
8. Use 2 μl of the reaction to transform 50 μl of competent cells. If reaction will not be used immediately for transformation, store at -20°C.

To learn more about Golden Gate Assembly, please visit www.neb.com/goldengate