(→Preparation of LB solid and liquid stock) |
(→Lab work) |
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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= | ||
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====Preparation of LB solid and liquid stock==== | ====Preparation of LB solid and liquid stock==== | ||
''By Caroline and Terrence'' | ''By Caroline and Terrence'' | ||
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- 1L of LB liquid : 20g/L powder LB + 1L MilliQ water <br/> | - 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 DS-SPcasN-, DS-TDcasN-, pZA21 and pZA31 ==== | + | ====Electrophoresis of PCR products DS-SPcasN-, DS-TDcasN-, pZA21 and pZA31==== |
''By Caroline'' | ''By Caroline'' | ||
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''By Mathilde '' | ''By Mathilde '' | ||
− | 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. | + | 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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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 + X-Gal 0.25μL/mL + IPTG 0.1 μL/mL) in duplicate . | + | 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