Team:Paris Saclay/Notebook/July/30
From 2013.igem.org
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==='''A - Aerobic/Anaerobic regulation system'''=== | ==='''A - Aerobic/Anaerobic regulation system'''=== | ||
- | ===='''Objective : obtaining | + | ===='''Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006'''==== |
===='''1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : NarK, NarG, NirB '''==== | ===='''1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : NarK, NarG, NirB '''==== | ||
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We used 30µL of DNA. | We used 30µL of DNA. | ||
- | ===='''Objective : obtaining | + | ===='''Objective : obtaining BBa_K1155003, BBa_K1155007'''==== |
- | ===='''1 - Tranformation of | + | ===='''1 - Tranformation of BBa_K1155003 in DH5α '''==== |
Abdou | Abdou | ||
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Protocol : [[Team:Paris_Saclay/Protocols/Bacterial transformation|Bacterial transformation]] | Protocol : [[Team:Paris_Saclay/Protocols/Bacterial transformation|Bacterial transformation]] | ||
- | ===='''2 - Digestion of | + | ===='''2 - Digestion of BBa_I732017 by EcoRI/PstI'''==== |
Zhou | Zhou | ||
Used quantities : | Used quantities : | ||
- | * | + | * BBa_I732017 : 10µL |
* Buffer FD : 3µL | * Buffer FD : 3µL | ||
* PstI FD : 1.5µL | * PstI FD : 1.5µL | ||
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We let the digestion 1h30 at 37°C. | We let the digestion 1h30 at 37°C. | ||
- | ===='''3 - Electrophoresis to check the digestion of | + | ===='''3 - Electrophoresis to check the digestion of BBa_I732017 by EcoRI/PstI'''==== |
Zhou | Zhou | ||
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|} | |} | ||
- | ===='''4 - Gel purification of digestion of | + | ===='''4 - Gel purification of digestion of BBa_I732017 by EcoRI/PstI'''==== |
Xavier | Xavier | ||
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|} | |} | ||
- | ===='''5 - Digestion of | + | ===='''5 - Digestion of BBa_I732017 by EcoRI/PstI'''==== |
Abdou | Abdou | ||
Used quantities : | Used quantities : | ||
- | * | + | * BBa_I732017 : 10µL |
* Buffer FD : 2µL | * Buffer FD : 2µL | ||
* SpeI FD : 1µL | * SpeI FD : 1µL | ||
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We let the digestion 1h30 at 37°C. | We let the digestion 1h30 at 37°C. | ||
- | ===='''6 - Electrophoresis to check the digestion of | + | ===='''6 - Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI'''==== |
Zhou | Zhou | ||
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| style="width:350px;border:1px solid black;vertical-align:top;" | | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
* Well 1 : ... | * Well 1 : ... | ||
- | * Well 2 : ... 20µL of | + | * Well 2 : ... 20µL of BBa_I732017 digested by EcoRI/SpeI+4µL of 6X loading dye |
* Gel : 1% | * Gel : 1% | ||
|} | |} | ||
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|} | |} | ||
- | ===='''7 - Gel purification of digestion of | + | ===='''7 - Gel purification of digestion of BBa_I732017 by EcoRI/PstI'''==== |
Xavier | Xavier |
Revision as of 09:34, 30 September 2013
Notebook : July 30
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : NarK, NarG, NirB
Xavier
Protocol : Ethanol precipitation
We used 30µL of DNA.
Objective : obtaining BBa_K1155003, BBa_K1155007
1 - Tranformation of BBa_K1155003 in DH5α
Abdou
Protocol : Bacterial transformation
2 - Digestion of BBa_I732017 by EcoRI/PstI
Zhou
Used quantities :
- BBa_I732017 : 10µL
- Buffer FD : 3µL
- PstI FD : 1.5µL
- EcoRI FD : 1.5µL
- H2O : 14µL
We let the digestion 1h30 at 37°C.
3 - Electrophoresis to check the digestion of BBa_I732017 by EcoRI/PstI
Zhou
File:Ps.jpg|350px]] |
|
Expected size :
- RBS-LacZ :
We obtain fragments at the right size. We will purify it. |
4 - Gel purification of digestion of BBa_I732017 by EcoRI/PstI
Xavier
Protocol : Gel purification
We used wrong enzymes. We will do it again with EcoRI/SpeI. |
5 - Digestion of BBa_I732017 by EcoRI/PstI
Abdou
Used quantities :
- BBa_I732017 : 10µL
- Buffer FD : 2µL
- SpeI FD : 1µL
- EcoRI FD : 1µL
- H2O : 6µL
We let the digestion 1h30 at 37°C.
6 - Electrophoresis to check the digestion of BBa_I732017 by EcoRI/SpeI
Zhou
File:Ps.jpg|350px]] |
|
Expected size :
- RBS-LacZ :
We obtain fragments at the right size. We will purify it. |
7 - Gel purification of digestion of BBa_I732017 by EcoRI/PstI
Xavier
Protocol : Gel purification
We lost our fragments. We will do the digestion again. |
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2 proteins
1 - PCR of : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I
Abdou, Xavier
Used quantities :
- Bphr2 Part I :
- Oligo 54F : 4µL
- Oligo 55R : 4µL
- Buffer Dream Taq : 40µL
- DNA of Pseudomonas pseudoalcaligenes : 2µL
- dNTP : 4µL
- Dream Taq : 1µL
- H2O : 146µL
- Bphr2 Part II :
- Oligo 56F : 4µL
- Oligo 57R : 4µL
- Buffer Dream Taq : 40µL
- DNA Pseudomonas pseudoalcaligenes : 2µL
- dNTP : 4µL
- Dream Taq : 1µL
- H2O : 146µL
- RBS-Bphr2 Part I :
- Oligo 58F : 4µL
- Oligo 57R : 4µL
- Buffer Dream Taq : 40µL
- DNA Pseudomonas pseudoalcaligenes : 2µL
- dNTP : 4µL
- Dream Taq : 1µL
- H2O : 146µL
- FNR Part I :
- Oligo 59F : 4µL
- Oligo 60R : 4µL
- Buffer Dream Taq : 40µL
- DNA Escherichia coli : 2µL
- dNTP : 4µL
- Dream taq : 1µL
- H2O : 146µL
- FNR Part II :
- Oligo 61F : 4µL
- Oligo 62R : 4µL
- Buffer Dream Taq : 40µL
- DNA Escherichia coli : 2µL
- dNTP : 4µL
- Dream Taq : 1µL
- H2O : 146µL
- RBS-FNR Part I :
- Oligo 63F : 4µL
- Oligo 62R : 4µL
- Buffer Dream Taq : 40µL
- DNA Escherichia coli : 2µL
- dNTP : 4µL
- Dream Taq : 1µL
- H2O : 146µL
- PSB1C3 :
- Prefixe ... : 4µL
- Suffixe ... : 4µL
- Buffer Dream Taq : 40µL
- DNA ... : 2µL
- dNTP : 4µL
- Dream Taq : 1µL
- H2O : 146µL
PCR Program :
- BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :
- FNR Part I, FNR Part II, RBS-FNR Part I :
- PSB1C3 :
2 - Electrophoresis of the PCR products : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I
File:Ps.jpg|350px]] |
|
Expected size
- BphR2 Part I : 178 kb
- BphR2 Part II : 790 kb
- RBS-BphR2 Part I : 197 kb
- FNR Part I : 597 kb
- FNR Part II : 200 kb
- RBS-FNR Part I : 615 kb
- PSB1C3 : ...
We can't see RBS-BphR2 Part I fragments at the good size. We will make the PCR again with a new PCR Program. We obtain BphR2 Part I, BphR2 Part II, FNR Part I, Fnr Part II and RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it. |
3 - PCR of : RBS-BphR2 Part I
Abdou, Xavier
Used quantities :
- Oligo 54F : 4µL
- Oligo 55R : 4µL
- Buffer .... : 40µL
- DNA : 2µL
- dNTP : 4µL
- .... : 1µL
- H2O : 146µL
PCR Program :
4 - Gel purification of PCR of : RBS-BphR2 Part I
Xavier
Protocol : Gel purification
We lost our fragments. We will do the PCR again. |
B - PCB sensor system
Objective : obtaining Bba_K1155002
1 - Colony PCR of Bba_K1155002 in DH5α
Abdou, Xavier
The transformation was good. We will make a Colony PCR. |
We used 25 different colonies.
Used quantities :
- Oligo 43F : 0.5µL
- Oligo 44R : 0.5µL
- Buffer Dream Taq : 35µL
- DNA : 2µL
- dNTP : 0.5µL
- Dream Taq : ... cf cahier !!!!!!!
- H2O : ... cf cahier !!!!!
2 - Electrophoresis of the Colony PCR of Bba_K1155002 in DH5α
Abdou, Xavier
File:Ps.jpg|350px]] |
|
Expected sizes :
- Bba_K1155002 : ...
We obtain fragments at the right size. We will sequence it. |
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