Golden Gate Assembly Protocol for Using NEBridge® Golden Gate Assembly Kit (BsaI-HF®v2) (E1601)
1. Set up assembly reactions as follows:
| REAGENT | NEGATIVE CONTROL | ASSEMBLY REACTION |
|---|---|---|
| pGGAselect Destination Plasmid*, 75 ng/μl | 1 μl | 1 μl |
| Inserts (user provided): - if precloned** - if in amplicon form*** |
– |
75 ng each plasmid |
| 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.
Videos
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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.
