Summary
- 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
|
gg2 pSB1A3 |
1.0
|
gg3 hPCD |
1.0
|
BL01 |
1.0
|
10x ligase buffer |
1.0
|
NEB T4 lgase |
0.25
|
BsmBI |
0.5
|
dH2O |
5.25
|
Total |
10.0
|
Could not perform bacterial transformation because SOC medium was in freezer. Vi performed BsmBI/T4 ligase and bacterial transformation for BL01 and hPCD and there were no colonies on plates.
|