08/29/13
- Golden Gate Assembly optimization - increase ligase amount; redo last successful hPCD assembly
Golden Gate
- Try to increase robustness by using more T4 ligase...
- hPCD + BL01 + pSB1A3
- pSB1A3 alone (neg ctrl)
- GG-PCR fragments were generated on 1/10/13 and diluted to 20 fmol/μL
- Try the Promega formula home-made buffer (worked last time)
- Golden Gate assembly reactions
1, 2. 0.25 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3
1, 2. 1.0 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3
Reagent |
Original protocol |
Original protocol |
More ligase |
More ligase
|
gg2 pSB1A3* |
1.0 |
1.0 |
1.0 |
1.0
|
gg3 hPCD* |
1.0 |
--- |
1.0 |
---
|
gg4 BL01* |
1.0 |
--- |
1.0 |
---
|
10x Promega ligase buffer |
1.0 |
1.0 |
1.0 |
1.0
|
NEB T4 ligase |
0.25 |
0.25 |
1.0 |
1.0
|
NEB BsmBI |
0.5 |
0.5 |
0.5 |
0.5
|
dH2O |
5.25 |
7.25 |
5.25 |
7.25
|
|
10.0 |
10.0 |
10.0 |
10.0
|
Note: *DNA is 20 fmol/μL
GG Thermal cycling
- [45°C, 2 min.; 16°C, 5 min.] x25
- 60°C, 20 min.
- 80°C, 20 min.
- 4°C, ∞
- Transformations
- 50 μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar
|