Team:Paris Saclay/Notebook/August/21
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Colonies repiquée dans 10µL d'eau. | Colonies repiquée dans 10µL d'eau. |
Revision as of 19:27, 20 September 2013
Notebook : August 21
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006
1 - Colony PCR of NarK, NarG or NirB with RBS-LacZ-Term or RBS-AmilCP-Term in PSB1C3 in DH5α
Damir, Xiaojing
The transformation was good. We will do a PCR Colony. |
Colonies repiquée dans 10µL d'eau. We do 8 PCR for each ligation.
Used quantities :
- Mix A :
- Buffer Dream Taq : 250µL
- dNTP : 50µL
- Oligo 44 : 50µL
- Dream Taq : 20µL
- H2O : 1.88mL
- NirB with RBS-LacZ-Term in PSB1C3 :
- 45µL of Mix A+1µL of oligo 45+2µL of DNA
- NirB with RBS-Amil CP-Term in PSB1C3 :
- 45µL of Mix A+1µL of oligo 45 +2µL of DNA
- NirB with RBS-LacZ-Term in PSB1C3 :
- NarG with RBS-LacZ-Term in PSB1C3 :
- 45µL of Mix A+1µL of oligo 41+2µL of DNA
- NarG with RBS-Amil CP-Term in PSB1C3 :
- 45µL of Mix A+1µL of oligo 41 +2µL of DNA
- NarG with RBS-LacZ-Term in PSB1C3 :
- NarK with RBS-LacZ-Term in PSB1C3 :
- 45µL of Mix A+1µL of oligo 47+2µL of DNA
- NarK with RBS-Amil CP-Term in PSB1C3 :
- 45µL of Mix A+1µL of oligo 47 +2µL of DNA
- NarK with RBS-LacZ-Term in PSB1C3 :
- Mix B :
- Mix A : 1.125mL
- Oligo 43 : 25µL
- NirB with RBS-LacZ-Term in PSB1C3 :
- 23µL of Mix B+2µL of DNA
- NirB with RBS-Amil CP-Term in PSB1C3 :
- 23µL of Mix B+2µL of DNA
- NirB with RBS-LacZ-Term in PSB1C3 :
- NarG with RBS-LacZ-Term in PSB1C3 :
- 23µL of Mix B+2µL of DNA
- NarG with RBS-Amil CP-Term in PSB1C3 :
- 23µL of Mix B+2µL of DNA
- NarG with RBS-LacZ-Term in PSB1C3 :
- NarK with RBS-LacZ-Term in PSB1C3 :
- 23µL of Mix B+2µL of DNA
- NarK with RBS-Amil CP-Term in PSB1C3 :
- 23µL of Mix B+2µL of DNA
- NarK with RBS-LacZ-Term in PSB1C3 :
PCR Program for NirB with RBS-LacZ-Term in PSB1C3, NarG with RBS-LacZ-Term in PSB1C3, NarK with RBS-LacZ-Term in PSB1C3 :
PCR Program for NirB with RBS-Amil CP-Term in PSB1C3, NarG with RBS-Amil CP-Term in PSB1C3, NarK with RBS-Amil CP-Term in PSB1C3 :
((((((((====3 - Culture ==== Nguyen
- BBa_K115500 strains (FNR repressor)in 5ml LB with Chlorenphenicol
- strain MG1665 Δ fnr:km clone1 in 5ml LB with kanamicine
Incubate at 37°C over night with shake 180 rpm.
Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3
1 - Electrophoresis of the digestion of Bba_J04450 by EcoRI/PstI
XiaoJing
[[]] |
|
Expected sizes :
- PSB3K3 : 2750 bp
- GFP : ... NON VISIBLE SUR LE GEL ???????
We obtain a fragment at the right size. We can purify it. |
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2 proteins
1 - Transformation of RBS-BphR2, FNR, RBS-FNR in DH5α
XiaoJing
Protocol : Bacterial transformation