Golden Gate Assembly Protocol for Using NEB Golden Gate Assembly Mix (E1600)

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***
75–100 ng each plasmid
2:1 molar ratio
(insert vector; pGGA = 2,174 bp)
NEB Golden Gate Buffer (10X) 2 μl 2 μl
NEB Golden Gate Assembly Mix 1 μl 1 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.

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 1-4 Inserts 37°C, 1 hr → 55°C, 5 min
For 5-10 Inserts (37°C, 1 min → 16°C, 1 min) x 30 → 55°C, 5 min
For 11-20 Inserts (37°C, 5 min → 16°C, 5 min) x 30 → 55°C, 5 min

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