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Team:Paris Saclay/Notebook/August/9
From 2013.igem.org
(Difference between revisions)
(→1 - Extraction of BBa_K115007 from DH5α) |
(→1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification) |
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*Well 1 : 6µL DNA Ladder | *Well 1 : 6µL DNA Ladder | ||
| - | *Well 2 : 5µL of BphR2 Part I+1µl of 6X loading dye | + | *Well 2 : 5µL of BphR2 Part I + 1µl of 6X loading dye |
| - | *Well 3 : 5µL of BphR2 Part II+1µl of 6X loading dye | + | *Well 3 : 5µL of BphR2 Part II + 1µl of 6X loading dye |
| - | *Well 4 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye | + | *Well 4 : 5µL of RBS-BphR2 Part I + 1µl of 6X loading dye |
| - | *Well 5 : 5µL of FNR Part I+1µl of 6X loading dye | + | *Well 5 : 5µL of FNR Part I + 1µl of 6X loading dye |
| - | *Well 6 : 5µL of FRN Part II+1µl of 6X loading dye | + | *Well 6 : 5µL of FRN Part II + 1µl of 6X loading dye |
| - | *Well 7: 5µL of RBS-FNR Part I+1µl of 6X loading dye | + | *Well 7: 5µL of RBS-FNR Part I + 1µl of 6X loading dye |
*Gel : 0.8% | *Gel : 0.8% | ||
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Revision as of 19:21, 3 October 2013
Notebook : August 9
Lab work
A - Aerobic/Anaerobic regulation system
Objective : characterize BBa_K1155000, BBa_K1155004, BBa_K1155005, BBa_K1155006
1 - Tranduction of Km in MG1655Z1
Abdou, Anaïs, Damir, Nadia, XiaoJing
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We observed lysis areas. We will continue the transduction protocol. |
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Picture: lysed cells comparison.
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Protocol : Transduction
Our Mutant bacteria was called BW : Δfnr::Km. Our wild type bacteria was called MG1655Z1. We used kanamycine antibiotic.
Objective : obtaining BBa_K1155007
1 - Extraction of BBa_K115007 from DH5αstrain
Abdou
Protocol : High-copy plamid extraction
We extracted plamid from colonies number 10, 14 and 15.
Nanodrop
- BBa_K1155007 in clone 10 : 38ng/µl
- BBa_K1155007 in clone 14 : 48.5ng/µl
- BBa_K1155007 in clone 15 : 52 ng/µl
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The extraction was good. We will sequence our plasmids. |
A - Aerobic/Anaerobic regulation system / B - PCB sensing system
Objective : Obtaining FNR and BphR2 proteins
1 - Electrophoresis of the PCR of BphR2 Part I, BphR2 Part II, RBS_BphR2 Part I, FNR Part I, FNR Part II, RBS_FNR Part I to check the gel purification
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Expected size :
- BphR2 Part I : 178 bp
- BphR2 Part II : 790bp
- RBS-BphR2 Part I : 197bp
- FNR Part I : 597 bp
- FNR Part II : 200bp
- RBS-FNR PartI : 615bp
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We lost all our PCR fragments. We will do the PCR again. |
2 - PCR of BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I
Anaïs, Damir, Nadia, XiaoJing
Used quantities :
- Bphr2 Part I :
- Oligo 54F : 1µL
- Oligo 55R : 1µL
- Buffer Phusion : 10µL
- DNA of Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL
- Bphr2 Part II :
- Oligo 56F : 1µL
- Oligo 57R : 1µL
- Buffer Phusion : 10µL
- DNA Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL
- RBS-Bphr2 Part I :
- Oligo 58F : 1µL
- Oligo 57R : 1µL
- Buffer Phusion : 10µL
- DNA Pseudomonas pseudoalcaligenes : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL
- FNR Part I :
- Oligo 59F : 1µL
- Oligo 60R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL
- FNR Part II :
- Oligo 61F : 1µL
- Oligo 62R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL
- RBS-FNR Part I :
- Oligo 63F : 1µL
- Oligo 62R : 1µL
- Buffer Phusion : 10µL
- DNA Escherichia coli : 1µL
- dNTP : 1µL
- Phusion : 0.5µL
- H2O : 35.5µL
PCR Program :
- BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :
- FNR Part I, FNR Part II, RBS-FNR Part I :
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