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Team:Paris Saclay/Notebook/August/5
From 2013.igem.org
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| - | * Well 1 | + | * Well 1 : 6µL of DNA Ladder |
| - | + | * Well 2 : 30µL of BBa_K1155003 from clone 9 digested by EcoRI/PstI+6µl of 6X loading dye | |
| - | * Well | + | * Well 3 : 5µL of BBa_K1155003 from clone 9+1µl of 6X loading dye |
| - | * Well | + | * Well 4 : 30µL of BBa_K1155003 from clone 11 digested by EcoRI/PstI+6µl of 6X loading dye |
| - | * Well | + | * Well 5 : 5µL of BBa_K1155003 from clone 11+1µl of 6X loading dye |
| - | * Well | + | * Well 6 : 30µL of BBa_K1155003 from clone 12 digested by EcoRI/PstI+6µl of 6X loading dye |
| - | * Well | + | * Well 7 : 5µL of BBa_K1155003 from clone 12+1µl of 6X loading dye |
| - | * Well | + | |
* Gel : 1% | * Gel : 1% | ||
|} | |} | ||
Revision as of 21:51, 1 October 2013
Notebook : August 5
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining BBa_K1155003
1 - Digestion of BBa_K1155003 by EcoRI/PstI to check the ligation between RBS-Amil CP,Term and PSB1C3
Nadia, XiaoJing
Used quantities :
- BBa_K1155003 : 5µL
- EcoRI FD : 1µL
- PstI FD : 1µL
- Buffer FD : 3µL
- H2O : 20µL
We let the digestion at 37°C during 15 minutes.
2 - Electrophoresis of the digestion of BBa_K1155003
Damir
| File:Ps.jpg|350px]] |
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Expected sizes :
- RBS_Amil CP-Term :
- PSB1C3 : 2070bp
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We obtain fragments at the right size. We will sequence it. |
Objective : obtaining BBa_K1155007
1 - Digestion of BBa_I732017 by EcoRI/SpeI
Abdou, Damir, Nadia,
Used quantities :
- BBa_I732017 : 41µL
- Buffer FD : 5µL
- EcoRI : 2µL
- SpeI : 2µL
We let the digestion at 1h30 at 37°C.
2 -Electrophoresis of the digestion of BBa_I732017 by EcoRI/SpeI
Damir, Nadia
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Expected sizes :
- RBS-LacZ : 3093 bp
- PSB1A2 : 2079 bp
|
We obtain fragments at the right size. We will make an elector-elution to extract RBS-LacZ. |
3 - PCR of PSB1C3
Nadia, XiaoJing
Used quantities :
- PSB1C3 : 2µL
- Buffer phusion : 10µL
- Oligo 64 : 2.5µL
- Oligo 65 : 2.5µL
- dNTP : 1µL
- Phusion : 0.25µL
- H2O : 36.75µL
PCR program :
Hybridation temperature gradient :
A - 65 B - 64.7 C - 64.1 D - 63.1 E - 62 F - 61.2 G - 60.5 H - 60
3 - Electrophoresis of PCR of PSB1C3
Nadia, XiaoJing
| [[]] |
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Expected sizes :
- PSB1C3 : 2070bp
|
We obtain fragments at the right size. We will purify it. |
4 - Gel purification of electrophoresis of PCR of PSB1C3
Nadia, XiaoJing
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Colony PCR of BBa_K1155004, BBa_K115005, BBa_K1155006 in DH5α
Damir, Nadia, XiaoJing
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Transformation of 07/31/13 works. We will do a PCR Colony. |
We mix our DNA in 10µL of H2O.
Used quantities :
- DNA : 2µL
- Mix : (it was divided in 25 tubes for 25 different colonies for each promotor with 23µL of mix in each tube)
- Oligo 44 : 3.5µL
- Oligo 45 : 3.5µL
- Buffer Dream Taq : 70µL
- dNTP : 28µL
- Dream Taq : 5µL
- H2O : 590µL
PCR Program :
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