Difference between revisions of "Team:Paris Saclay/Notebook/July/12"
(→High fidelity PCR on bacteria transformed with pPS16_004 and pPS16_007) |
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
=Tuesday 12<sup>th</sup> July= | =Tuesday 12<sup>th</sup> July= | ||
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===Bringing DNA closer=== | ===Bringing DNA closer=== | ||
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''By Caroline'' | ''By Caroline'' | ||
| − | Plasmids extracted the 4/07/16 were used to amplify the dCas9 needed for the | + | Plasmids extracted the 4/07/16 were used to amplify the dCas9 needed for the bring DNA closer tool and the pZA21 plasmid in which the tool will be inserted. A PCR was carried out using Q5<sup>®</sup> High-Fidelity 2X Master Mix and following the [[Team:Paris_Saclay/Experiments#Q5PCR|usual protocol]] adapted to have 50µL at the end. |
====Electrophoresis of the PCR amplification==== | ====Electrophoresis of the PCR amplification==== | ||
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5µL of the amplifications are mixed with 1µL of purple loading dye. The solutions are put into a 0.8% agarose gel and migrated for 30min. | 5µL of the amplifications are mixed with 1µL of purple loading dye. The solutions are put into a 0.8% agarose gel and migrated for 30min. | ||
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[[File:T--Paris_Saclay--160712_bringingDNAcloser_échelle.JPG|400px|thumb|right|Migration of SP, TD and pZA21]] | [[File:T--Paris_Saclay--160712_bringingDNAcloser_échelle.JPG|400px|thumb|right|Migration of SP, TD and pZA21]] | ||
| − | ====Liquid culture of transformed NM, TD, ST1 and SP | + | ====Liquid culture of transformed NM, TD, ST1 and SP dcas9==== |
''By Mathilde and Laetitia'' | ''By Mathilde and Laetitia'' | ||
| − | Two colonies from each | + | Two colonies from each Petri dishe was incubated in 1mL of LB and Spectinomycin (50µg/mL). |
Our 8 cultures were put in incubation at 37°c, 180 rpm ON. | Our 8 cultures were put in incubation at 37°c, 180 rpm ON. | ||
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Cells transformed on 11/07/16 grew overnight. However no colony was observed. | Cells transformed on 11/07/16 grew overnight. However no colony was observed. | ||
| − | + | In order to control if there is any bacteria transformed but not among what was spread on Petri dish, we redo the experiment. 100 µL (500µL concentrated) of transformed bacteria for each condition, conserved at -4°C from yesterday, were split in 3 solutions : | |
* 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) | * 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) | ||
* 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) | * 30µL of bacteria + 3mL LB + Streptomycin (50µg/mL) | ||
* 30µL of bacteria + 3mL LB + Chloramphenicol (30µg/mL) | * 30µL of bacteria + 3mL LB + Chloramphenicol (30µg/mL) | ||
| − | Cells were incubated for 8h at 37°C, | + | Cells were incubated for 8h at 37°C, 200rpm. |
No bacteria grew so we concluded that there was a problem with the transformation. | No bacteria grew so we concluded that there was a problem with the transformation. | ||
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===Visualization=== | ===Visualization=== | ||
| − | ====Transformation of | + | ====Transformation of gBlocks 4.2 pPS16_008 ==== |
''By Laetitia and Mathilde'' | ''By Laetitia and Mathilde'' | ||
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* 20mL of LB+Agar | * 20mL of LB+Agar | ||
| − | Cells transformed with the two gBlocks constructions were | + | Cells transformed with the two gBlocks constructions were spread on Petri dishes in duplicata (50µL and 150µL) and a control construct was also spread. |
The plates were put in incubation at 37°c overnight. | The plates were put in incubation at 37°c overnight. | ||
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''By Alice'' | ''By Alice'' | ||
| − | A PCR with Q5® High-Fidelity 2X Master Mix was performed on clones selected after screening PCR of the [[Team:Paris_Saclay/Notebook/July/11#Colony screening PCR pPS16_004 and pPS16_0076|11/07/16]], following [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]].[[Team:Paris_Saclay/Experiments#primers|1151_pheoR and 1152_pheoF]] primers were used. Annealing temperature was 60°C | + | A PCR with Q5® High-Fidelity 2X Master Mix was performed on clones selected after screening PCR of the [[Team:Paris_Saclay/Notebook/July/11#Colony screening PCR pPS16_004 and pPS16_0076|11/07/16]], following [[Team:Paris_Saclay/Experiments#Q5PCR|this protocol]].[[Team:Paris_Saclay/Experiments#primers|1151_pheoR and 1152_pheoF]] primers were used. Annealing temperature was 60°C. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put on gel. PCR products were migrated 30min at 100V. |
PCR products expected were : | PCR products expected were : | ||
Latest revision as of 15:19, 9 October 2016
