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Team:Paris Saclay/Notebook/August/29
From 2013.igem.org
Contents |
Notebook : August 29
summary
- We got clonies on ligation promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3
promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3 promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3t so we do Colony PCR for it
- We didn't get clonies on Gibson so we do a gel electrophoresis of parts of Gibson assembly to verify the size
- Digestion Dnp1 purification of the the PSB1C3 clean for Gibson and electrophoresis of it .
lab work
- A.aero/anaerobic regulation system
- Digestion for PSB3K3
| DNA | 9µl |
| Ecoil I | 2µl |
| PST I | 2µl |
| H2O | 5µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_LacZ+Term_10(08/09/13)
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_LacZ+Term_11(08/09/13)
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_LacZ+Term_15(08/09/13)
B1-8: promoter fnr(activator)nirB plus RBS_AmilCP+Term_PSB1C3
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_AmilCP+Term_9(02/09/13)
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Digestion for RBS_AmilCP+Term_12(02/09/13)
| DNA | 14µl |
| xpe I | 2µl |
| PST I | 2µl |
| buffer | 2µl |
| total | 20µl |
- Degestion product quantification
- We used the nano drop to measure the DNA in 260nm and we found its concentration
| name | fnr(repressor)dig EcoilI and speI | fnr nark dig EcoilI and speI |
| conc. | 25.0ng/µl | 13.6ng/µl |
2 -Gel electrophoresis of Degestion product of promoter fnr(activator).
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3 -Gel electrophoresis of Degestion product of RBS_AmilCP+Term and RBS_LacZ+Term.
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1 - Colony PCR of Gibson assembly.
Colonies repiquée dans 10µL d'eau. We do 8 PCR for each ligation.
PCR MIX for 55 tubes:
- Buffer Dream Taq : 137.5µL
- dNTP : 27.5µL
- Oligo 44 : 27.5µL
- Oligo 43: 27.5µL
- Dream Taq : 11µL
- H2O : 1.144mL
Add 2µL bacteria into 23µL PCR MIX in PCR tube PCR Program
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