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Karmella's BioBrick Cloning
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01/15/13
- Golden gate assembly: test different ligase buffers
Golden Gate Assembly optimization
Buffer |
Tris-HCl |
MgCl2 |
DTT |
ATP
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10x Promega |
300 mM (pH 7.8) |
100 mM |
100 mM |
10 mM
|
10x NEB |
500 mM (pH 7.5) |
100 mM |
100 mM |
10 mM
|
2x Roche |
? |
? |
? |
?
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- Make Promega buffer from scratch.
- Golden Gate assembly reactions
1, 2. Promega - hPCD+BL01+pSB1A3
3, 4. Promega - pSB1A3
5, 6. NEB - hPCD+BL01+pSB1A3
7, 8. NEB - pSB1A3
9, 10. Roche - hPCD+BL01+pSB1A3
11, 12. Roche - pSB1A3
Reagent |
Promega (1,2) |
Promega (3, 4) |
NEB (5, 6) |
NEB (7, 8) |
Roche (9, 10) |
Roche (11, 12)
|
gg2 pSB1A3* |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
gg3 hPCD* |
1.0 |
--- |
1.0 |
--- |
1.0 |
---
|
gg4 BL01* |
1.0 |
--- |
1.0 |
--- |
1.0 |
---
|
ligase buffer |
1.0 |
1.0 |
1.0 |
1.0 |
5.0 |
5.0
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NEB T4 ligase |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
0.25
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NEB BsmBI |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5
|
dH2O |
5.25 |
7.25 |
5.25 |
7.25 |
1.25 |
3.25
|
|
10.0 |
10.0 |
10.0 |
10.0 |
10.0 |
10.0
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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
- All odd samples: 50 μL DH5α-Turbo; ice 5 min.; plate on amp agar
- All even samples: 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
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