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Team:Paris Saclay/Notebook/July/26
From 2013.igem.org
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(→1 - Denaturation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP) |
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* Well 1 : 5µL of NirB+1µL of 6X loading dye | * Well 1 : 5µL of NirB+1µL of 6X loading dye | ||
| - | * Well 2 : 5µL of NirB+1µL of 6X loading dye | + | * Well 2 : 5µL of NirB+1µL of 6X loading dye |
* Well 3 : 5µL of NarG+1µL of 6X loading dye | * Well 3 : 5µL of NarG+1µL of 6X loading dye | ||
* Well 4 : 5µL of NarG+1µL of 6X loading dye | * Well 4 : 5µL of NarG+1µL of 6X loading dye | ||
* Well 5 : 6µL DNA Ladder | * Well 5 : 6µL DNA Ladder | ||
| - | * Well 6 : 5µL of NarK+1µL of 6X loading dye | + | * Well 6 : 5µL of NarK+1µL of 6X loading dye |
* Well 7 : 5µL of NarK+1µL of 6X loading dye | * Well 7 : 5µL of NarK+1µL of 6X loading dye | ||
* Well 8 : 5µL of NarK+1µL of 6X loading dye | * Well 8 : 5µL of NarK+1µL of 6X loading dye | ||
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Expected sizes : | Expected sizes : | ||
| - | * NarK, NarG, NirB : | + | * NarK, NarG, NirB : 200bp |
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Revision as of 15:49, 4 October 2013
Notebook : July 26
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining BBa_K1155003
1 - Inactivation of EcoRI/SpeI used for the digestion of PCR products : RBS-AmilCP
Abdou
Protocol : Ethanol precipitation
We used 90µL of DNA. At the end, we mix our DNA with 20µL of water.
2 - Electrophoresis of the digestion of PCR products : RBS-AmilCP
Abdou
| [[]] |
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Expected sizes :
- RBS-AmilCP :
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We obtain fragments at the right size. We will ligate RBS-AmilCP with Term. |
3 - Ligation of RBS-AmilCP and Term-PSB1C3
Xavier
Used quantities :
- RBS-AmilCP : 2µL
- Term in PSB1C3 : 2.5µL
- Buffer : 1µL
- Ligase : 1µL
- H2O : 3.5µL
Objective : obtaining BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Electrophoresis of PCR products : NarK, NarG, NirB
Abdou
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Expected sizes :
- NarK, NarG, NirB : 200bp
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We lost all our PCR products. We will do PCR again using more concentrated DNA and oligos. |
2 - PCR of NarK, NarG, NirB
Abdou, Xavier
Used quantities :
- NarK :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo ... : 2µL
- Oligo ... : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
- NarG :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo ... : 2µL
- Oligo ... : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
- NirB :
- Buffer phusion : 10µL
- dNTP : 1µL
- Oligo ... : 2µL
- Oligo ... : 2µL
- Concentrated DNA : 2µL
- Phusion : 0.25µL
PCR Program :
3 - Electrophoresis of PCR products : NarK, NarG, NirB
Abdou, Xavier
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|
Expected sizes :
- NarK, NarG, NirB : ...
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We obtain fragments at the right size. We can purify it. |
4 - Gel purification of PCR products : NarK, NarG, NirB
Xavier
Protocol : [http://www.mn-net.com/tabid/1452/default.aspx Gel purification ]
At the end we mix our DNA in 20µL of H2O.
5 - Digestion of PCR products : NarK, NarG, NirB by EcoRI/PstI
Abdou
Quantities used :
- NarK, NarG, NirB : 10µL
- Buffer FD : 3µL
- EcoRI FD : 1.5µL
- PstI FD : 1.5µL
- H2O : 14µL
We let the digestion at 37°C during 1h30 and then at -20°C.
B - PCB sensor system
Objective : obtaining BBa_K1155002
1 - Tranformation of BBa_K1155002 in DH5α
Abdou, Xavier
Protocol : Bacterial tranformation
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