Template:IONIS Paris/Notebook/11-09-15
Digestion
| Tube | VVD YC | VVD YN |
|---|---|---|
| H2O MQ | 1 μL | 1 μL |
| Buffer 2.1 | 2 μL | 2 μL |
| DNA | 15 μL | 15 μL |
| Enzyme 1 | EcoRI 1 μL | XbaI 1 μL |
| Enzyme 2 | SpeI 1 μL | PstI 1 μL |
37°C, 1h heat kill: 65°C, 20min
Expected results
Results
Ligation
| Tube | pSB1C3-VVD YC | pSB1C3-VVD YN | pSB1C3 |
|---|---|---|---|
| H2O MQ | 2,2 μL | 3,4 μL | 1,8 μL |
| T4 ligase buffer | 1 μL | 1 μL | 2 μL |
| Insert | VVD YN, 4,1 µL | VVD YC, 2,9 µL | pDawn (28/07), 7,2 µL |
| Backbone | 2,2 μL | 2,2 μL | 8 μL |
| T4 ligase | 0,5 μL | 0,5 μL | 1 μL |
37°C, 30min heat kill: 65°C, 20min
- Transformation of competent cells with pSB1C3-VVD YC + VVD YN and pSB1C3-VVD YN + VVD YC (1 µL or 4 µL have been used); 30°C all the weekend long
Ligation pDawn Gblock
| Tube | pDawn I-II-III | pDawn IV-V |
|---|---|---|
| T4 ligase buffer | 2 μL | 2,3 μL |
| pDawn I | 2,3 μL | - |
| pDawn II | 12 μL | - |
| pDawn III | 2,7 μL | - |
| pDawn IV | - | 13,2 μL |
| pDawn V | - | 6,2 μL |
| T4 ligase | 1 μL | 1 μL |
Room temperature, 45min heat kill: 65°C, 20min
Purification of pSB1C3-pDawn
20 µL of ligation product + 80 µL of H2O MQ
Add 1/10 (10 µL) of the volume of NaOAc 3M pH 5.2
Add 2,5 volumes (275 µL) of Ethanol 100%
Mix and stored at -20°C for 1 hour at least
Centrifuge 30 sec, 14000 rpm at 4°C
Discard the supernatant
Resuspend the pellet with 500 µL of ethanol 70%, invert up the tube several time
Centrifuge 5 min, 14000 rpm at 4°C
Discard the supernatant and let dry the pellet
Resuspend the pellet with the right amount of water following the final concentration expected (20 µL here)
Ligation pDawn Gblock
| Tube | pDawn I-II-III-IV-V |
|---|---|
| pDawn I-II-III | 20 μL |
| pDawn IV-V | 22,3 μL |
| T4 ligase | 1 μL |
Room temperature, 45min heat kill: 65°C, 20min
11 Sept. 15