Difference between revisions of "Team:Pasteur Paris/Week 14"

 
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Latest revision as of 02:18, 19 September 2015

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 14

08/31 - 09/04




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

  2. Co-transfection in yeast of nicked pRS415 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 nicked pRS415 as vector no colonies has grown on Petri dishes.

  4. Assembly of cluster 1:
    • XbaI and PstI enzymatic digestion.
    • Gel migration on 1% agarose gel.
    • Gel Extraction following the Macherey-Nagel protocol.
    • DNA Concentration

    5. 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

  5. MidiPrep of BBa_K808030:
    • SpeI 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.

    6. 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

  6. Ligation of BBa_K808007 and BBa_K808030.

  7. Transformation in electro-competent DH5-α cells.

  8. Preparation for sequencing.

  9. Assembly of cluster 2 (BBa_K808007 + BBa_K808030).

  10. PCR amplification of BBa_K936011 and BBa_K936023 using Q5 High fidelity Master Mix.


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