Team:Bordeaux/NotebookAugust22

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Latest revision as of 02:16, 18 October 2014

22nd August

Molecular biology

  • Chewback : purification with Qiagen Kit, dosage on nanodrop
    ELP (6240pb) 8,2 ng/μl
    RFP (706pb) 12,8ng/μl

    We want to use 25 fmol => we use promega website (μg to pmol converter)
    ELP = 0,002pmol/μl => 25 fmol = 12,5μl
    RFP = 0,027pmol/μl => 25 fmol = 0,9μl

    Do the mix on ice :
    RFP 0,9μl
    Plasmid 12,5μl
    Master Mix 12,5μl

    Inoculate immediately 1h at 50°C
    • Transformation of competent cells and spread on LB+Amp plates
    • Gel 1% (30min, 100V)

  • Biobrick assembly
    • Digestion
      • Dilution ELP 2,1μl +7,7μl H2O
      Dilution SLP 3,6μl +6,4μl H2O
      Dilution RLP 1,97μl +8,03μl H2O

      incubation 1h30 at 37°C :
      Enzyme 1 (Nhe1 or X) 0,5μl
      Enzyme 2 (S) 0,5lμl
      10X Buffer 4 5μl
      DNA 10μl
      H2O qsp 50μl
      - Enzyme inactivated 20min at 80°C
      - Gel 1% (30min, 100V)

    • Ligation
      • Plasmid 4μl
      Insert 4μl
      Buffer 10X 1μl
      Taq ligase 0,5μl
      H2O qsp 10μl
      => 30min at 16°C

    • Transformation


Purification

  • We change the water of dialysis at 9h.

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