Difference between revisions of "Golden Gate"
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For a total of 15 μl reaction, while working on ice: | For a total of 15 μl reaction, while working on ice: | ||
* mix 1 μl of each plasmid (each at concentration 100ng/μl) | * mix 1 μl of each plasmid (each at concentration 100ng/μl) | ||
| − | * add 0.5 μl BsaI ( | + | * add 0.5 μl BsaI (5 units) |
* add 0.75 μl T4 ligase (NEB) | * add 0.75 μl T4 ligase (NEB) | ||
* add 1.5 μl ligase buffer (avoid freeze-thaw cycles to protect the ATP) | * add 1.5 μl ligase buffer (avoid freeze-thaw cycles to protect the ATP) | ||
| − | * | + | * add 0.15 μl of bovine serum albumin (10 mg/ml) |
Incubate in a thermocycler using 'GoldenGate' program: | Incubate in a thermocycler using 'GoldenGate' program: | ||
| Line 16: | Line 16: | ||
!Duration | !Duration | ||
|- | |- | ||
| − | | | + | |40X || 37°C || 2 min |
|- | |- | ||
| || 16°C || 5 min | | || 16°C || 5 min | ||
Revision as of 13:55, 10 December 2018
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 units)
- add 0.75 μl T4 ligase (NEB)
- add 1.5 μl ligase buffer (avoid freeze-thaw cycles to protect the ATP)
- add 0.15 μl of bovine serum albumin (10 mg/ml)
Incubate in a thermocycler using 'GoldenGate' program:
| Number of cycles | Temperature | Duration |
|---|---|---|
| 40X | 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.
