Template:IONIS Paris/Notebook/26-08-15
Digestion of pSB1C3 (from iGEM) and miniprep
| Tube | pSB1C3 | pSB1C3 | Miniprep |
|---|---|---|---|
| Water | 6 µL | 12 µL | 12 µL |
| Buffer 2.1 | 4 µL | 2 µL | 2 µL |
| Plasmid | 8 µL | 5 µL | 5 µL |
| Enzyme EcoRI | 1 µL | - | 0,5 µL |
| Enzyme PstI | 1 µL | - | 0,5 µL |
| Enzyme XbaI | - | 0,5 µL | - |
| Enzyme SpeI | - | 0,5 µL | - |
37°C, 1h
Expected results
Results
There are only bands corresponding to linearized pSB1C3 and one band, unexpected, corresponding to unknown sequence.
PCR of pDawn (1,2,3,4) and VVD-YN (1,2,1/4)
| pDawn (1,2,3,4) | VVD-YN (1,2,1/4) | |
|---|---|---|
| MQ Water | 40 µL | 40 µL |
| RB Buffer | 5 µL | 5 µL |
| Mg2+ | 1,5 µL | 1,5 µL |
| dNTP 10 µM | 1 µL | 1 µL |
| Primer Fwd 50 µM | 0,5µL pDawn Fwd I | 0,5µL VVD Fwd I |
| Primer Rev 50 µM | 0,5µL pDawn Rev II | 0,5µL YN155 Rev II |
| DNA | 1µL | 1µL |
| Taq Pol enzyme | 0,5 µL | 0,5 µL |
Ligation of Gblocks into pSB1C3
| Tube | VVD-YC155 | VVD-YN155 | GFP rev | H/E rev | CCDB rev | HOKD rev |
|---|---|---|---|---|---|---|
| Water | 2,4 µL | 1,3 µL | 2,2 µL | - | 3,5 µL | 4,5 µL |
| T4 ligase Buffer | 1 µL | 1 µL | 1 µL | 1 µL | 1 µL | 1 µL |
| Gblock | 3,6 µL | 4,7 µL | 3,8 µL | 5,8 µL | 2,5 µL | 1,5 µL |
| pSB1C3 | 2,5 µL | 2,5 µL | 2,5 µL | 2,5 µL | 2,5 µL | 2,5 µL |
| T4 ligase | 0,5 µL | 0,5 µL | 0,5 µL | 0,5 µL | 0,5 µL | 0,5 µL |
Room temperature, 30min
Ligation of pSB1C3 (from iGEM) XbaI-SpeI (circularization)
| Tube | pSB1C3 |
|---|---|
| Water | 3,5 µL |
| T4 ligase Buffer | 1 µL |
| Plasmid | 5 µL |
| T4 ligase | 0,5 µL |
Room temperature, 1h
Expected results
Results
Same band for each sample Impossible Fail
Expected results
Results
Very soft bands at 2000 bp (pSB1C3) and 4000 bp (???)
Some bands between them (apparent on the screen of the gel reader, not on the picture…)
Transformation of bacteria with ligation products
26 August 15