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.
|
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
|
Bacterial transformation
- Added total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
- Incubated on ice for 2 min., heat shock at 42°C for exactly 45 sec., and placed on ice.
- Added 800 μL sterile SOC medium.
- Grow with shaking at 37°C for 30 min.(Taped tubes to the shaker rack.)
- Pelleted the cells at top speed in a microcentrifuge for 3 min. at room temperature.
- Discarded the supernatant. Resuspended the cells in 100 μL LB + antibiotic.
- Plate cells on pre-warmed LB agar + antibiotic. (I plated 100 μL of the cells. Then put beads on plates and shook.) Protocol didn't specify.) Grow overnight at 37°C.
- Check results tomorrow.
There were no colonies on the plates. It's possible that the concentration of the ligation product is to low, so I will try to amplify.
|
