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.

KuceraBecky

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.