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Team:Paris Saclay/Notebook/August/29
From 2013.igem.org
Notebook : August 29
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize Bba_K1155000 and Bba_K1155004, Bba_K1155005, Bba_K1155006
1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions
XiaoJing
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Purification of 08/28/13 didn't work. We have blue colonies for Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic and anaerobic conditions. We also have blue colonies for NirB with RBS-LacZ-Term in PSB1C3 in anaerobic conditions. We will streak these colonies again. |
We streak :
- Pfnr with RBS-Amil CP-Term in PSB1C3 with O2 at 37°C
- Pfnr with RBS-Amil CP-Term in PSB1C3 without O2 at 37°C
- Pfnr with RBS-Amil CP-Term in PSB1C3 with O2 at 30°C
- Pfnr with RBS-Amil CP-Term in PSB1C3 without O2 at 30°C
- NirB with RBS-LacZ-Term in PSB1C3 with O2 with Xgal at 37°C
- NirB with RBS-LacZ-Term in PSB1C3 without O2 with Xgal at 37°C
We also purify Pfnr with RBS-Amil CP-Term in PSB1C3 in liquid culture at 37°C using :
- Pfnr with RBS-Amil CP-Term in PSB1C3 in aerobic conditions :
- LB : 10 mL
- Clone : ...
- Pfnr with RBS-Amil CP-Term in PSB1C3 in anaerobic conditions :
- LB : 50mL
- Clone : ...
2 - Digestion of Bba_K1155000, Bba_K1155003, Bba_K1155007 by Xbal/PstI
XiaoJing
We used clone 9 and 12 for Bba_K1155003, clone 10, 11 and 15 for Bba_K1155007
Used quantities :
- Bba_K1155003, Bba_K1155007
- DNA : 14µL
- Buffer FD : 2µL
- XbaI FD : 2µL
- PstI FD : 2µL
- Bba_K1155000 :
- DNA : 5µL
- Buffer FD : 2µL
- SpeI FD : 2µL
- PstI FD : 2µL
- H2O : 9µL
We let the digestion 30 minutes at 37°C.
3 - Electrophoresis to check the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI
XiaoJing
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Expected sizes :
- RBS-LacZ-Term :
- RBS-Amil CP-Term :
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Expected sizes :
- Pfnr :
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We obtain fragments at the right size. We will purify them. |
4 - Gel purification of the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI
XiaoJing
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Nanodrop :
- Pfnr : 26.2ng/µL
5 - Electrophoresis to check the gel purification of the digestion of Bba_K1155000 by PstI/SpeI, Bba_K1155003, Bba_K1155007 by Xbal/PstI
Damir
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Expected sizes :
- RBS-LacZ-Term :
- RBS-Amil CP-Term :
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We obtain fragments at the right size for RBS-LacZ-Term. We will ligate it. |
5 - Electrophoresis to check the digestion of Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI/PstI and plasmids already digested by SpeI and after digested by PstI
XiaoJing
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Expected sizes :
- NarK, NarG, NirB : ...
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We obtain fragments at the right size for NarK. We will ligate it |
((((((((((((((====Objective : obtaining Pfnr, NarK, NarG or NirB and RBS-LacZ-Term or RBS-AmilCP-Term in PSB3K3====
1 - Digestion of Bba_J04450 by EcoRI/PstI
Anaïs
Used quantities :
- Buffer FD: 2µL
- H2O : 5µL
- DNA : 9µL
- EcoRI FD : 1µL
- PstI FD : 1µL
We let the digestion at 37°C during 10 minutes ??????
2 - Electrophoresis to check the digestion of Bba_J04450 by EcoRI/PstI
XiaoJing
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expected sizes :
- PSB3K3 :
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We obtain fragments at the right size. We will ligate it. |
3 - Gel purification of the digestion of Bba_J04450 by PstI/SpeI
XiaoJing
Protocol : Gel purification
Nanodrop :
- Pfnr : 7.2ng/µL)))))))))))))))))))))))
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FRN and BphR2 proteins
1 - Colony PCR of FNR, RBS-FNR and RBS-BphR2 in DH5α
XiaoJing
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Transformation of 08/28/13 works. We will do a Colony PCR. |
COLONIES PIQUEES DANS 20µL d'eau pour chaque colonie.
Used quantities :
- DNA : 2µL
- Mix : (it was divided in 8 tubes for 8 different colonies for each assembly with 23µL of mix in each tube. We do it twice.)
- Oligo 43 : 27.5µL
- Oligo 44 : 27.5µL
- dNTP : 27.5µL
- Buffer Dream Taq : 137.5µL
- Dream Taq : 11µL
- H2O : 1144µL
