Team:Paris/August 8
From 2008.igem.org
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* Program PCR_Screening : Annealing 55°C - Time élongation 1'30" - Number cycle : 29 | * Program PCR_Screening : Annealing 55°C - Time élongation 1'30" - Number cycle : 29 | ||
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==== '''PCR verification/Analyse''' ==== | ==== '''PCR verification/Analyse''' ==== | ||
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* Incubate 2h30 at 37°C | * Incubate 2h30 at 37°C | ||
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==== Purification/Analysis ==== | ==== Purification/Analysis ==== | ||
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==> '''Conclusions : We validate the digestion of the vector and the insert'''. Now we can be sure, that we detect anything for '''FlhDC genes'''. | ==> '''Conclusions : We validate the digestion of the vector and the insert'''. Now we can be sure, that we detect anything for '''FlhDC genes'''. | ||
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== '''Transformation results''' == | == '''Transformation results''' == | ||
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== '''PCR Screening of Ligation Transformants'''== | == '''PCR Screening of Ligation Transformants'''== | ||
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* one toothpick of each clone's colony per tube | * one toothpick of each clone's colony per tube | ||
* Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29 | * Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29 | ||
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=== Conditions of electrophoresis === | === Conditions of electrophoresis === | ||
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|align="center"|1 | |align="center"|1 | ||
|align="center"|2 to 9 | |align="center"|2 to 9 | ||
- | |style="background: #cbff7B"|<center> | + | |align="center"|261 pb |
- | + | |style="background: #cbff7B"|<center>300 pb</center> | |
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|align="center"|PCR_125' | |align="center"|PCR_125' | ||
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|align="center"|1 | |align="center"|1 | ||
|align="center"|10 to 17 | |align="center"|10 to 17 | ||
- | |style="background: #cbff7B"|<center> | + | |align="center"|261 pb |
- | + | |style="background: #cbff7B"|<center>300 pb</center> | |
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|align="center"|PCR_126' | |align="center"|PCR_126' | ||
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|align="center"|2 | |align="center"|2 | ||
|align="center"|2 to 9 | |align="center"|2 to 9 | ||
- | |style="background: #cbff7B"|<center> | + | |align="center"|260 pb |
- | + | |style="background: #cbff7B"|<center>300 pb</center> | |
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|align="center"|PCR_127' | |align="center"|PCR_127' | ||
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|align="center"|2 | |align="center"|2 | ||
|align="center"|10 to 17 | |align="center"|10 to 17 | ||
- | |style="background: #ff6d73"|<center> | + | |align="center"|446 pb |
- | + | |style="background: #ff6d73"|<center>1,000 pb</center> | |
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Revision as of 15:48, 11 August 2008
Minipreps : Plasmid extractionExtraction of pSB3K3 et E0240 in pSB1A2 plasmid from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN) by QIACube.
Amplification of Genes of interest (OmpR, EnvZ, FlhDC)We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.
PCR amplificationProtocol
For each samples, 1 µl dNTP
PCR verification/AnalyseAfter the PCR :
==> Conclusion : we observed the size expected for the PCR products, but not for FlhDC gene.
Digestion of PCR productsProtocol :
Purification/AnalysisElectrophoresis
==> Conclusions : We validate the digestion of the vector and the insert. Now we can be sure, that we detect anything for FlhDC genes.
Transformation results
PCR Screening of Ligation TransformantsUse of 8 clones of Ligation transformants for PCR screening
Protocol of screening PCR
Conditions of electrophoresis
Results of electrophoresis
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