Golden Gate
From PombEvolution
Jump to navigationJump to search
Cut-ligation
Following Binder et al 2014.
BsaI cut-ligations
For a total of 15 μl reaction, while working on ice:
- mix 1 μl of each plasmid (each at concentration 100ng/μl)
- add 0.5 μl BsaI (5–10 units)
- add 0.75 μl T4 ligase (NEB)
- add 1.5 μl ligase buffer (avoid freeze-thaw cycles to protect the ATP)
- asdd BsaI cut-ligations 0.15 μl of bovine serum albumin (10 mg/ml)
Incubate in a thermocycler using 'GoldenGate' program:
| Number of cycles | Temperature | Duration |
|---|---|---|
| 30X | 37°C | 2 min |
| 16°C | 5 min | |
| 1X | 37°C | 5 min |
| 50°C | 5 min | |
| 80°C | 5 min | |
| 7°C | Inf |
[E_coli_Chemical_Transformations | Transform 3–5 μl into E. coli] TOP10 cells.
