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Team:Paris Saclay/Notebook/July/1
From 2013.igem.org
Notebook : July 1
summary
- started the Restriction digestion and ligation to get the BioBrick fnr into plasmid PSB1C3 by using restrictin ensyme EcoR I and PST I.
- Sent an E-mail to M. Nesbeth from UCL for requesting the Biobrick BBa_K239005 which is created by iGEM UCL team in 2009
- Designed oligopeptides for BphR2 (regulator, sensitive for PCBs), Transcriptional regulator of Pseudomonas oleovorans /pseudoalcaligenes group
lab work
- A.aero/anaerobic regulation system
- 1.BioBrick promotor fnr(repressor) in plasmid PSB1C3
- Digestion for fnr and PSB1C3
- 2 enzymes EcoR I and PST I can be used in one common buffer: orange buffer (10X).
- For PCR products:
| PCR products | 20µl |
| EcoR I | 0.75µl |
| PST I | 0.75µl |
| H2O | 5.5µl |
| buffer | 3µl |
| total | 30µl |
- For plasmid PSB1C3:
| Plasmid | 4µl |
| EcoR I | 0.5µl |
| PST I | 0.5µl |
| H2O | 2.2µl |
| buffer | 0.8µl |
| total | 8µl |
- Ligation
- After 3h of digestion, we mixed the digestion products:
| PCR product | 30µl |
| PSB1C3 | 4µl |
| Ligation buffer | 2µl |
| H2O | 14µl |
- Then we performed a precipitation by ethanol in order to inactivate the enzymes. And suspended the deposit by:
| mixture | control |
| 2µl ligation buffer | 2µl ligation buffer |
| 1µl ligase T4 | 1µl ligase T4+2µl PSB1C3 |
| 17µl H2O | 15µl H2O |
- The incubation was during 1H30.
- B.PCBs sensor system
- 1.BioBrick BphR2(regulator) in plasmid PSB1C3
- 1.BioBrick BphR2(regulator) in plasmid PSB1C3
- Using software gene manager to find the oligopeptide for amplification of BphR2.
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