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February 9, 2013
Golden Gate Assembly
- Mediated assembly
- The volume of purified DNA (x) needed to dilute in a final volume of 20 μL = length in bp ÷ measured ng/μL * 20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng * 20 μL final volume
- Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20
| DNA |
length (bp) |
ng/μL |
vol. |
dH2O
|
| H2B |
410 |
77.995 |
1.4 |
18.6
|
| LOV |
461 |
80.443 |
1.5 |
18.5
|
| pSB1A3 |
2000 |
77.4 |
6.7 |
13.3
|
| Reagent |
Assembly |
Control
|
| 20 fmol H2B |
1.0 |
0
|
| 20 fmol LOV |
1.0 |
0
|
| 20 fmol pSB1A3 |
1.0 |
1.0
|
| 10x T4 ligase buffer (Promega) |
1.0 |
1.0
|
| T4 ligase (NEB) |
0.25 |
0.25
|
| BsmBI |
0.5 |
0.5
|
| dH2O |
5.25 |
7.25
|
| Total vol. |
10.0 |
10.0
|
- Bacterial transformation
- Add total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
- Incubate on ice for 2 min., heat shock at 42°C for exactly 45 sec., immediately place on ice.
- Add 800 μL sterile SOC medium.
- Grow with shaking at 37°C for 30 min.
- Pellet the cells at top speed in a microcentrifuge for 3 min. at room temp.
- Discard the supernatant. Resuspend the cells in 100 μL LB + antibiotic.
- Plate cells on pre-warmed LB agar + antibiotic. Grow overnight at 37°C. Quick-transormation (e.g., DH5α-Turbo) is not recommended
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