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 Engineering PC-TFs
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Summary
- * Performed BsmBI/ T4 ligase mediated assembly
- BsmBI cuts the DNA fragments and creates complementary overhangs.
- Complementary sticky ends anneal via base pairing.
- T4 ligase seals gaps in the phosphodiester DNA backbone.
| Reagent
|
Vol.
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Thermal cycling
- [45°C, 2 min.; 16°C 5 min.] x25
- 60°C, 10 min.
- 80°C, 20 min.
- 4°C, ∞
|
| 20 fmol (1 μL) of each DNA part |
up to 8.0
|
| 10x T4 ligase buffer (Promega) |
1.0
|
| T4 ligase (NEB) |
0.25
|
| BsmBI |
0.5
|
| dH2O |
0.25
|
| |
10.0 μL
|
| Reagent
|
1
|
2
|
3
|
| gg2 pSB1A3 |
1.0 |
1.0 |
1.0
|
| gg3 hPCD |
1.0 |
1.0 |
1.0
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| BL02 |
1.0 |
--- |
---
|
| BL03 |
--- |
1.0 |
---
|
| BL04 |
--- |
--- |
1.0
|
| 10x ligase buffer |
1.0 |
1.0 |
1.0
|
| NEB T4 lgase |
0.25 |
0.25 |
1.0
|
| BsmBI |
0.5 |
0.5 |
0.5
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| dH2O |
5.25 |
5.25 |
5.25
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| Total |
10.0 |
10.0
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Engineering PC-TFs
