Difference between revisions of "Golden Gate"
From PombEvolution
Jump to navigationJump to search| Line 1: | Line 1: | ||
== Cut-ligation == | == Cut-ligation == | ||
| − | Following Binder et al 2014. | + | Following Binder et al 2014. and Kakui et al 2015. |
| + | |||
| + | Dilute each module element (including linear modules such as the GOI) in 20 fmol/μl: | ||
| + | * Measure the [[DNA concentration with the NanoDrop]] | ||
| + | * Obtain the length of the plasmid or fragment | ||
| + | * Calculate the dilution to obtain 13.3 ng/μl per 1-kilobase DNA (for a plasmid: insert length + 3.5kb) | ||
| + | * Make the dilutions | ||
=== BsaI cut-ligations === | === BsaI cut-ligations === | ||
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 | + | * mix 1 μl of each plasmid |
* add 0.5 μl BsaI (5 units) | * add 0.5 μl BsaI (5 units) | ||
* add 0.75 μl T4 ligase (NEB) | * add 0.75 μl T4 ligase (NEB) | ||
| Line 15: | Line 21: | ||
!Temperature | !Temperature | ||
!Duration | !Duration | ||
| + | |- | ||
| + | |1X || 37°C || 10 min | ||
|- | |- | ||
|40X || 37°C || 2 min | |40X || 37°C || 2 min | ||
Revision as of 14:11, 10 December 2018
Cut-ligation
Following Binder et al 2014. and Kakui et al 2015.
Dilute each module element (including linear modules such as the GOI) in 20 fmol/μl:
- Measure the DNA concentration with the NanoDrop
- Obtain the length of the plasmid or fragment
- Calculate the dilution to obtain 13.3 ng/μl per 1-kilobase DNA (for a plasmid: insert length + 3.5kb)
- Make the dilutions
BsaI cut-ligations
For a total of 15 μl reaction, while working on ice:
- mix 1 μl of each plasmid
- 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 |
|---|---|---|
| 1X | 37°C | 10 min |
| 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.
