03/13/13
Golden Gate Assembly
- First, make fresh 10x Promega Buffer:
| Reagent |
Conc. |
Volume
|
| 1M Tris-HCl (pH 7.8) |
300 mM |
300.0 μL
|
| 1M MgCl2 |
100 mM |
100.0
|
| 1M DTT |
100 mM |
100.0
|
| ATP |
10 mM |
10.0
|
| dH2O |
|
490.0
|
| Final vol. |
|
1000 uL
|
- Golden Gate assembly reactions
- Promega - H2B + LOV-his + pSB1A3
- Promega - pSB1A3
- Roche - H2B + LOV-his + pSB1A3
- Roche - pSB1A3
| Reagent |
Promega (1) |
Promega (2) |
Roche (9, 10) |
Roche (11, 12)
|
| gg2 pSB1A3* |
1.0 |
1.0 |
1.0 |
1.0
|
| gg3 hPCD* |
1.0 |
--- |
1.0 |
---
|
| gg4 BL01* |
1.0 |
--- |
1.0 |
---
|
| ligase buffer |
1.0 |
1.0 |
5.0 |
5.0
|
| NEB T4 ligase |
0.25 |
0.25 |
0.25 |
0.25
|
| NEB BsmBI |
0.5 |
0.5 |
0.5 |
0.5
|
| dH2O |
5.25 |
7.25 |
1.25 |
3.25
|
| |
10.0 |
10.0 |
10.0 |
10.0
|
Note: *DNA is 20 fmole/μL
Thermal cycler
- [45°C, 2 min.; 16°C, 5 min.] x25
- 60°C, 20 min.
- 80°C, 20 min.
- 4°C, ∞
- Transformations
- 50 μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar
|
Project name
Main project page
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