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Team:Paris Saclay/Notebook/August/13
From 2013.igem.org
Notebook : August 13
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006
1 - Electrophoresis to check the digestion of Bba_K1155000 by SpeI/PstI
Nadia
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Expected sizes :
- Pfnr : ...
- PSB1C3 : ...
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We can't see any band for Bba_K1155000 digestion. The digestion failed because we used the wrong enzymes. We will do it again with the good ones. |
2 - Digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and by SpeI/EcoRI
Anaïs, Nadia
Used quantities :
- Bba_K1155000 :
- Buffer FD : 5µL
- H2O : 39µL
- DNA : 5µL
- SpeI FD : 1µL
- Bba_K1155000 :
- Buffer FD : 5µL
- H2O : 38µL
- DNA : 5µL
- SpeI FD : 1µL
- EcoRI FD : 1µL
- Bba_K1155004, Bba_K1155005, Bba_K1155006 :
- Buffer FD : 2µL
- H2O : 7µL
- DNA : 10µL
- SpeI FD : 1µL
- Bba_K1155004, Bba_K1155005, Bba_K1155006 :
- Buffer FD : 2µL
- H2O : 6µL
- DNA : 10µL
- SpeI FD : 1µL
- EcoRI FD : 1µL
We let digestions at 37°C during 10 minutes ??????
3 - Electrophoresis to check the digestion of Bba_K1155000, Bba_K1155004, Bba_K1155005, Bba_K1155006 by SpeI and by SpeI/EcoRI
Anaïs, Nadia
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Expected sizes :
- Pfnr : ...
- PSB1C3 : 2070 kb
- Pfnr in PSB1C3 : ...
- Bba_K1155000 : ...
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We lost all our digestion product so we will do it again. |
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Expected sizes :
- NarK, Nar G, NirB : 200kb
- PSB1C3 : ...
- NarK in PSB1C3, NarG in PSB1C3, NirB in PSB1C3 : ...
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We lost all our double digestion product so we will do it again. We obtain Bba_K1155004, Bba_K1155005, Bba_K1155006 digested by SpeI fragments at the right size. We can purify 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 : 6µL
- DNA : 10µL
- 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
Anaïs, Nadia
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Expected sizes :
- PSB3K3 : ...
- GFP : ...
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RESULTAT ???? MAUVAISE DIGESTION ????? APPAREMMENT NON CA SUITE LE 22/08 |
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2 proteins (Gibson assembly)
1 - PCR of FRN Part I, FNR Part II, BphR2 Part I
Anaïs
QUANTITES UTILISEES !!!!!!!!!!!!!!!!!!!!!!!!!!!!
PCR Program : SCHEMA !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
2 - Electrophoresis of PCR products : FRN Part I, FNR Part II, BphR2 Part I
Damir
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Expected sizes :
- FNR Part I : 597 kb
- FNR Part II : 200 kb
- BphR2 Part I : 178 kb
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It's impossible to read the first gel. We do it again. In the second gel, we obtain FNR Part I and FNR Part II fragments at the right size. We can purify it. We also obtain BphR2 Part I frangment at the right size but it was mix with other DNA frangments. We will try to make a gel purification of it. |
3 - Electrophoresis of PCR product : BphR2 Part I
Anaïs, Damir, Nadia
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Protocol : Gel purification
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Even if the two lightest stripes are overlapping, aims to do a second gel purification with these two stripes, we did the gel purification . After argumentation, we decided to do the PCR of BphR2 Part I again. AVEC DE NOUVELLES QUANTITES ????????????? ET UN NOUVEAU PROGRAMME : TEMPERATURE ANNEALING AUGMENTEE A 60°C ????????????????? |
4 - PCR of BphR2 Part I
Anaïs
Used quantities :
- Oligo 54F : 1µL
- Oligo 55R : 1µL
- DNA : 2µL
- Buffer Phusion : 10µL
- dNTP : 1µL
- Phusion : 1µL
- H2O : 33.5µL
PCR Program : SCHEMA !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
5 - Gel purification of PCR product : FNR Part I, FNR Part II, RBS-FNR Part I, BphR2 Part II, RBS-BphR2 Part I
XiaoJing
Protocol : Gel purification
Nanodrop :
- FNR Part I : 152.7ng/µL
- FNR Part II : 137.2ng/µL
- RBS-FNR Part I : 153.7ng/µL
- BphR2 Part II : 129.9ng/µL
- RBS-BphR2 Part I : 158.1ng/µL
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CONCLUSION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! |
