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 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.