Golden Gate Assembly Protocol for Using NEB Golden Gate Assembly Mix (E1600)
This protocol is for E1600 which has been discontinued and replaced with E1601. If you have questions or need assistance with E1600 please contact technical support.
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.
Videos
-
Restriction Enzymes in Golden Gate Assembly
Type IIS restriction enzymes have both recognition and binding sites, but cut downstream of the recognition site, creating 4-base overhangs ideal for re-assembly. View a list of TypeIIS enzymes.
