Golden Gate Assembly Protocol for Using NEB® Golden Gate Assembly Kit (BsaI-HF®v2) (E1601)

1. Set up assembly reactions as follows:

REAGENT NEGATIVE CONTROL ASSEMBLY REACTION
pGGA Destination Plasmid*, 75 ng/μl 1 μl 1 μl
Inserts (user provided):
- if precloned**
- if in amplicon form***
75 ng each plasmid
2:1 molar ratio
(insert : vector; pGGA = 2,174 bp; 75 ng = 0.056 pmol)
T4 DNA Ligase Buffer (10X) 2 μl 2 μl
NEB Golden Gate Assembly Mix 1 - 2 μl**** 1 - 2 μl
Nuclease-free H2O to 20 μl to 20 μl

* or user provided.
** Precloned inserts must possess BsaI restriction sites at both ends of the insert sequence and in the proper orientation.
*** Amplicon inserts must possess 5´ flanking bases and BsaI restriction sites at both ends of the amplicon and in the proper orientation.
**** For assemblies < 10 inserts, use 1 μl : for assemblies > 10 inserts, use 2 μl.


Note: Negative controls are not routinely done for assembly reactions, but are described for first time users.

2. Choose the appropriate assembly protocol

INSERT NUMBER SUGGESTED ASSEMBLY PROTOCOL
For 1 Insert 37°C,  5 min (cloning) or 37°, 1 hr (library preparation) → 60°C, 5 min
For 2-4 Inserts 37°C, 1 hr → 60°C, 5 min
For 5-10 Inserts (37°C, 1 min → 16°C, 1 min) x 30 → 60°C, 5 min
 For 11 - 20+ inserts (37°C, 5 min → 16°C, 5 min) x 30 → 60°C, 5 min

To learn more about NEB Golden Gate, please see our technical note.