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Team:Paris Saclay/Notebook/August/13
From 2013.igem.org
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Anaïs | Anaïs | ||
| - | QUANTITES UTILISEES | + | QUANTITES UTILISEES !!!!!!!!! |
PCR Program : | PCR Program : | ||
| - | SCHEMA | + | SCHEMA !!!!!!!!!!!!!! |
===='''2 - Electrophoresis of PCR products : FRN Part I, FNR Part II, BphR2 Part I'''==== | ===='''2 - Electrophoresis of PCR products : FRN Part I, FNR Part II, BphR2 Part I'''==== | ||
Revision as of 20:51, 10 September 2013
Notebook : August 13
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining ...
1 - Electrophoresis to check the digestion of Bba_K1155000 by SpeI/PstI
Nadia
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Expected sizes :
- Pfnr : ...
- PSB1C3 : ...
|
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
- 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
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 : ...
|
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 : ...
|
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. |
4 - Digestion of Bba_J04450 by EcoRI/PstI
Anaïs
- Buffer FD: 2µL
- H2O : 6µL
- DNA : 10µL
- EcoRI FD : 1µL
- PstI FD : 1µL
5 - Electrophoresis to check the digestion of Bba_J04450 by EcoRI/PstI
Anaïs, Nadia
| [[]] |
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Expected sizes :
- PSB3K3 : ...
- GFP : ...
|
RESULTAT ???? MAUVAISE DIGESTION ????? |
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2 proteins
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
|
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 frangment at the right size but it was mix with other DNA frangments. We will try to make a gel purification of it. |
3 - Gel purification of PCR product : BphR2 Part I
Anaïs, Damir, Nadia
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Protocol : Gel purification
|
Even if the two lightest stripes are overlapping, aims to do a second gel purifiacation with these two stripes, we did the gel purification . After argumentation, we decided to do the PCR of bphR2 Part I again. |
4 - PCR of BphR2 Part I
Anaïs
Protocol : 08/09/13
New quantities :
- Oligo 54F : 1µL
- Oligo 55R : 1µL
- DNA : 2µL
- Buffer Phusion : 10µL
- dNTP : 1µL
- Phusion : 1µL
- H2O : 33.5µL
