Difference between revisions of "Team:Paris Saclay/Notebook/July/18"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
=Monday 18<sup>th</sup> July= | =Monday 18<sup>th</sup> July= | ||
| − | + | ||
====Preparation of LB solid and liquid stock==== | ====Preparation of LB solid and liquid stock==== | ||
''By Caroline and Terrence'' | ''By Caroline and Terrence'' | ||
| − | - 1L of LB liquid : 20g/L powder LB + 1L water | + | - 1L of LB liquid : 20g/L powder LB + 1L MilliQ water <br/> |
- 500mL of LB solid : 500mL of LB liquid + 7.5g/L of Agar<br/> | - 500mL of LB solid : 500mL of LB liquid + 7.5g/L of Agar<br/> | ||
| − | The | + | The solutions were put in autoclave for sterilization. |
| − | === | + | ===Bringing DNA closer=== |
| − | ==== Electrophoresis of PCR products | + | ====Electrophoresis of PCR products DS-SPcasN-, DS-TDcasN-, pZA21 and pZA31==== |
''By Caroline'' | ''By Caroline'' | ||
| − | PCR products obtained the 13/07/2016 were put to migrate for 30min in a 0 | + | PCR products obtained the 13/07/2016 were put to migrate for 30min in a 0.8% agarose gel. |
| + | |||
| + | [[File:T--Paris_Saclay--160718_bringingDNAcloser_échelle1.JPG|400px|thumb|right|Migration of SP,TD, pZA21 and pZA31]] | ||
===Biobrick characterization=== | ===Biobrick characterization=== | ||
| − | ====LB culture of electro-transformed BL21 cells [[Team:Paris_Saclay/Notebook/July/13#Biobrick_characterization| | + | ====LB culture of electro-transformed BL21 cells ([[Team:Paris_Saclay/Notebook/July/13#Biobrick_characterization|cf. protocol]])==== |
''By Mathilde '' | ''By Mathilde '' | ||
| − | Each transformation : | + | Each transformation: BL21|pcl_TAA + K1372001, BL21|pcl_TAG + K1372001 and BL21|pcl_Tq + K1372001 was added to 3mL of LB and one or two antibiotics. |
Three conditions were tested for each transformation : | Three conditions were tested for each transformation : | ||
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* 15µL of Chloramphenicol (30µg/mL) + 5 µL of Streptomycin (50µg/mL) | * 15µL of Chloramphenicol (30µg/mL) + 5 µL of Streptomycin (50µg/mL) | ||
| − | Cultures were put in incubation at | + | Cultures were put in incubation at 37°C, 180 rpm. |
===Visualization=== | ===Visualization=== | ||
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''By Caroline'' | ''By Caroline'' | ||
| − | PCR products obtained the [[Team:Paris_Saclay/Notebook/July/13#Amplification of DS_SPcasN, DS_TDcasN, pZA21 and pZA31 by Phusion PCR|13/07/2016]] were put to migrate for 30min in a 0 | + | PCR products obtained the [[Team:Paris_Saclay/Notebook/July/13#Amplification of DS_SPcasN, DS_TDcasN, pZA21 and pZA31 by Phusion PCR|13/07/2016]] were put to migrate for 30min in a 0.8% agarose gel |
| − | + | ||
====Transformation of bacteria with [[Team:Paris_Saclay/Notebook/July/4#pPS16_004|pPS16_004]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_007|pPS16_007]] ==== | ====Transformation of bacteria with [[Team:Paris_Saclay/Notebook/July/4#pPS16_004|pPS16_004]] and [[Team:Paris_Saclay/Notebook/June/28#pPS16_007|pPS16_007]] ==== | ||
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PCR made on bacteria transformed with pPS16_004 and pPS16_007 did not give expected results. We supposed that bacteria used for these PCRs did not still have the plasmid of interest. That is why we performed another transformation of DH5α competent cells with pPS16_004 and pPS16_007. We transformed cells following [[Team:Paris_Saclay/Experiments#Heat_shock_competent_cells|this protocol]]. We add 5μl of sterile H<SUB>2</SUB>O to the ligation product rest and used the whole for the transformation. | PCR made on bacteria transformed with pPS16_004 and pPS16_007 did not give expected results. We supposed that bacteria used for these PCRs did not still have the plasmid of interest. That is why we performed another transformation of DH5α competent cells with pPS16_004 and pPS16_007. We transformed cells following [[Team:Paris_Saclay/Experiments#Heat_shock_competent_cells|this protocol]]. We add 5μl of sterile H<SUB>2</SUB>O to the ligation product rest and used the whole for the transformation. | ||
| − | Finally, we spread cells on Petri dishes (LB + Amp 50μg/mL + | + | Finally, we spread the cells on Petri dishes (LB + Amp 50μg/mL + X-Gal 0.25μL/mL + IPTG 0.1 μL/mL) in duplicate . |
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 15:34, 9 October 2016
