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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
|
| 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)
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| 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 |
---
|
| 10x ligase buffer |
1.0 |
1.0
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| NEB T4 lgase |
0.25 |
0.25
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| BsmBI |
0.5 |
0.5
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| dH2O |
5.25 |
7.25
|
| |
10.0 |
10.0
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Thermal cycler
- [45°C, 2 min.; 16°C, 5 min.] x25
- 60°C, 20 min.
- 80°C, 20 min.
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Karmella's BioBrick Cloning
