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.
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.
