Team:Pasteur Paris/Week 14

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Week 14

08/31 - 09/04




  1. After 72h of incubation, only contamination was observed

  2. Co-transfection in yeast of nicked pRS415 nicked and Cre with extensions the protocol given by Heloise MÜLLER

  3. After Four days of culture of the electroporated yeast BY4742 with Cre+ homologous extension and the plasmid pRS415 nicked as vector no colonies has grown on Petri dishes.

  4. Assembly of cluster 1:

    • Xba I and Pst I enzymatic digestion.
    • Gel migration on 1% agarose gel.
    • Gel Extraction following the Macherey-Nagel protocol
    • DNA Concentration

    Dilution 1/25

    DNA Concentration (ng/µl)

    OD (230 nm)

    OD (260 nm)

    OD (280 nm)

    OD (260 nm) / OD (230 nm)

    BBa_K808030

    0.200

    0.674

    0.040

    0.000

    0.010

    BBa_K808031

    0.200

    0.560

    0.003

    0.000

    0.010

    1. Midiprep of BBa_K808030:
      • Spe I and PstI enzymatic digest of BBa_K808007 with the phosphatase step.
      • Gel migration on 1% agarose gel.
      • Gel Purification following the Macherey-Nagel protocol
      • DNA concentration assay using a spectrophotometer.

      2. Dilution 1/25

      DNA Concentration (ng/µl)

      OD (230 nm)

      OD (260 nm)

      OD (280 nm)

      OD (260 nm) / OD (230 nm)

      OD (260 nm) / OD (280 nm)

      808007

      0.800

      0.070

      0.017

      0.014

      0.240

      1.250

      808007

      1,100

      0,133

      0.022

      0.017

      0.170

      1.280

      1. Ligation of BBa_K808007 and BBa_K808030
      2. Transformation in electro-competent DH5-⍺ cells.
      3. Preparation for sequencing.

      4. Assembly of cluster 2:
      5. PCR amplification of BBa_K936011 and BBa_K936023 using Q5 High fidelity Master Mix.

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