2/2/2013
- Perform 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.
|
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
|
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
|
dH2O |
5.25 |
5.25 |
5.25
|
Total |
10.0 |
10.0
|
|