1/29/13
Type IIS Assembly Day 2
Digestion/ ligation reaction
Dilute the purified PCR product to 20 fmol/μL- Measure ng/μL of the purified sample.
- The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * length in bp * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ measured ng/μL
- Formula: x = length in bp ÷ measured ng/μL * 0.26
Purified PCR Product to 20fmol/μL
| Sample
|
μL of Sample
|
μL of dH2O
|
| 1. pSB1A3 |
7.8 |
12.2
|
| 2. hPCD |
2.8 |
17.2
|
| 3. BL02 |
6.4 |
13.6
|
| 4. BL03 |
5.5 |
14.5
|
| 5. BL04 |
8.8 |
11.2
|
| 6. fshPCD |
2.3 |
7.7
|
- 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
|
| |
10.0 |
10.0
|
|
Engineering PC-TFs
