NEBridge™ Ligase Master Mix Protocol Guidelines

This table provides guidance on using NEBridge™ Ligase Master Mix (MM) with NEB Type IIS restriction enzymes for Golden Gate Assembly. Recommendations differ based on the number of fragments to be assembled as well as the choice of Type IIS restriction enzyme. All incubations should be followed by an end soak for 5 min at 60°C prior to transformation. Store reactions at -20°C if not immediately being used for transformations.

 

2 fragments* 3-6 fragments 7+ fragments**
5 µl NEBridge Ligase MM
15 µl rxn volume
15-60 min 37°C ***
-or-
15-30 cycles of 42°C X 1 min, 16°C X 1 min****
End soak 60°C X 5 min

5 µl NEBridge Ligase MM 
15 µl rxn volume
30 cycles of 37°C X 1 min, 16°C X 1 min***
-or-
30 cycles of 42°C X 1 min, 16°C X 1 min****
End soak 60°C X 5 min

10 µl NEBridge Ligase MM
30 µl rxn volume
30-60 cycles of 37°C X 5 min, 16°C X 5 min***
-or-
30-60 cycles of 42°C X 5 min, 16°C X 5 min****
End soak 60°C X 5 min

BbsI-HF® 1 µl  (20U) 1 µl (20U) 1 µl (50U)a
BsaI-HFv2 1 µl (20U) 1 µl (20U) 1 µl (20U)
BsmBI-v2 3 µlb (30U) 3 µl(30U) 6 µlb (60U)
BspQI 1 µl (10U) 1 µl (10U) 2 µl (20U)
Esp3I 2 µl (20U) 3 µl (30U) 4 µl (40U)
PaqCI® 1 µlc (10U) 1 µlc (10U) 2.5 µlc (25U)
SapI 1 µl (10U) 1 µl (10U) 2 µl (20U)

* For 2 fragment assembly, 15 min or 15 cycles for single insert cloning; 60 min or 30 cycles for library construction
** For 7+ fragment assembly, 30 cycles for 7-13 fragment assembly; 60 cycles for 14+ fragment assembly 
*** Reaction protocol for BbsI-HF, BsaI-HFv2, Esp3I, PaqCI and SapI
**** Reaction protocol for BsmBI-v2 and BspQI. Optimum reaction temperature is 42°C rather than 37°C.

a = Requires use of NEB #R3539M (50U/μl)
b = Use of less enzyme will reduce performance
c = Recommended PaqCI Activator : PaqCI ratio is 1:1 (pmol:U). Use 0.5 µl of PaqCI Activator (20 µM) for 2 and 3-6 fragments; 1.25 µl of PaqCI Activator (20 µM) for 7+ fragments