Difference between revisions of "Team:Paris Saclay/Notebook/July/11"

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''By Alice''
 
''By Alice''
  
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The colony screening PCR of the [[Team:Paris_Saclay/Notebook/July/8#Colony screening PCR|08/07/16]] is performed again. After transformation, only white bacteria are selected (blue white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1152_pheoF and 1151 5_pheoR) upstream and downstream  the insertion site are chosen. . PCR products expected below :
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The colony screening PCR of the [[Team:Paris_Saclay/Notebook/July/8#Colony_screening_PCR|08/07/16]] is performed again. After transformation, only white bacteria are selected (blue white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1152_pheoF and 1151 5_pheoR) upstream and downstream  the insertion site are chosen. . PCR products expected below :
  
 
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Revision as of 15:46, 12 July 2016

Monday 11th July

Lab work

Biobrick characterization

Electrocompetents and transformation by electroporation of BL21

By Charlène

BL21 cells were grown up to OD600nm=7.14. They were then made competent and transformed with K1372001 extracted from clone 2 and pcl_TAA, pcl_TAG or pcl_Tq to create the following strains:

  • BL21|K1372001+pcl_TAA
  • BL21|K1372001+pcl_TAG
  • BL21|K1372001+pcl_Tq


Bringing DNA closer

DH5α transformation with DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN-

By Laetitia and Mathilde

DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- plasmids were resuspended with 5μL of sterilized water. 5 tubes were prepared :

  • one control tube with only 50μL of DH5α heat-shock competent cells
  • four tubes with 50μL of DH5α heat-shock competent cells + 1μL of plasmid

The regular heat-shock competent cells transformation protocol was used.

Transformed cells were plated in duplicata (10μL and 50μL) on 8 Petri dishes (spectinomycin 50μg/mL). Control cells were plated on another plate (spectinomycin 50μg/mL).

Visualization

Colony screening PCR on bacteria transformed with pPS16_004 and pPS16_007

By Alice

The colony screening PCR of the 08/07/16 is performed again. After transformation, only white bacteria are selected (blue white screen). They are expected to have plasmids with inserts of interest. PCR with DreamTaq DNA Polymerase is performed following this protocol. Clones selected for PCR are kept on LB+Amp+XGal(2µLXGal/1000µLH2O)+IPTG(2µLIPTG/1000µLH2O). Two primers (1152_pheoF and 1151 5_pheoR) upstream and downstream the insertion site are chosen. . PCR products expected below :

Plasmids Band size (bp)
pPS16_004 846
pPS16_007 744