Golden Gate Assembly Protocol - Dueber
Updated 5/17/2021 01:43 PM
Introduction
Materials
- DNA insert/plasmid
- T4 DNA Ligase buffer
- NEB
- T7 DNA Ligase
- NEB
- BsaI or BsmBI restriction enzyme
- Both 10,000U/mL from NEB
Procedure
- Prepare a reaction mixture:
Golden Gate Reaction Mixture
Item Item | Volume (uL) Volume (uL) | |
1 1 | Total Volume: | 10 |
2 2 | Each DNA insert/plasmid | 0.5 |
3 3 | T4 DNA Ligase buffer | 1 |
4 4 | T7 DNA Ligase | 0.5 |
5 5 | BsaI or BsmBI | 0.5 |
6 6 | Water | 7.5 |
The amount of DNA inserts can optionally be normalized to equimolar concentrations (~20fmol each) to improve assembly efficiencies
- Incubate reaction mixture in a thermocycler according to the following program:
25 cycles of digestion and ligation (42oC for 2 min, 16oC for 5 min) followed by...
a final digestion step (60oC for 10 min) and...
a heat inactivation step (80oC for 10 min).
In some cases, you can omit the final digestion and heat inactivation steps.
Lifted from Dueber et. al. and modified for our own use.