"
Team:Paris Saclay/Notebook/August/26
From 2014.igem.org
Contents |
Tuesday 26th August
Lab work
plasmid extraction : pGEMTeasy + chromoprotein
by Laetitia
We used the bacteria containing pGEMTeasy + chromoprotein launched by Laetitia monday the 25th for the plasmid extraction, 6 independent cultures which come from the 6 stocks made yesterday.
To extract the plasmid, we used the plasmid DNA purification kit (Macherey-Nagel). Protocol for low copy plasmid.
Digestion of pGEMTeasy+chromoproteinby XbaI and PstI
by Laetitia
The goal is to check the presence of the insert inside the plasmid. We digest the 6 samples of purified plasmid.
| component | volume |
|---|---|
| Plasmid | 10 μl |
| Fast Digest buffer 10X | 1,5 μl |
| XbaI | 0.5μl |
| PstI | 0.5μl |
| H20 | 2,5 μl |
1h at 37°C
Electrophoresis of the digestion product of pGEMTeasy+chromoprotein
by Laetitia
T : control negatif without enzyme
| component | volume |
|---|---|
| blue | 2 μl |
| Plasmid | 2 μl |
| H20 | 8μl |
D : sample digested by XbaI and PstI
D1 - D2 - D3 - D4 - D5 - D6 - T - L
D - Lemon Scent
Streaking of colonies transformed by BBa_K762100+pGEMTeasy (LS)
Due to yesterday's strange results, we conduct 30 streakings in a petri dish: 28 white colonies and 2 blue colonies.
Digestion of pGEMTeasy+ LS by SacI
The goal is to check if the limonen synthase gene is really inside the plasmid. We used the two samples named " LS2 " and " LS3 " which are normally pGEMTeasy+LS .
| component | volume |
|---|---|
| Plasmid | 20 μl |
| Fast Digest buffer 10X | 3μl |
| SacI | 1μl |
| H20 | 6μl |
Electrophoresis of the digestion products by SacI
T : control negatif without enzyme
| component | volume |
|---|---|
| blue | 2 μl |
| Plasmid | 2 μl |
| H20 | 8μl |
D : sample digested by SacI
L - TLS2 - DLS2 - TLS3 - DLS3
