Team:Heidelberg/Notebook/Sensing Group/Notebook/8thweek

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Monday, 09/22/2008

  • test digestion of F1_LuxP_NdeI (F1 cloned in pDK48 with NdeI), F2_LuxP_NdeI, F1_LuxP_KpnI (F1 cloned in pDK48 with KpnI) and F2_LuxP_KpnI (F2 cloned in pDK48 with KpnI) with NcoI (NEBuffer 3)
Digestions of Fusion constructs with NcoI. Positive clones show two bands at 4308 and 2915 bp
Digestions of Fusion constructs with NcoI. Positive clones show two bands at 4308 and 2915 bp
  • Transformation of Fusion constructs (F1(NdeI)_LuxP_pDK48, F1(KpnI)_LuxP_pDK48 and F2(NdeI)_LuxP_pDK48) into MG1655 and HCB33 cells
  • Preparation of cultures for Maxipreps of Fusion constructs

Tuesday, 09/23/2008

  • Maxiprep of LuxP-Fusion constructs (F1(NdeI)_LuxP_pDK48, F1(KpnI)_LuxP_pDK48 and F2(NdeI)_LuxP_pDK48)
  • Cloning of Fusion_LuxP in pBAD33 with BamHI/PstI
  • digestions of Fusion constructs and pBAD33 as described in gel image descriptions
Digestion of Fusion constructs and pBAD33 with BamHI/PstI for subsequent cloning.
Digestion of Fusion constructs and pBAD33 with BamHI/PstI for subsequent cloning.
Digestion of Fusion constructs with NcoI and PstI to check for correct insert and Maxiprep digetions.
Digestion of Fusion constructs with NcoI and PstI to check for correct insert and Maxiprep digetions.


  • O/N culture of Fusion-LuxP-pDK48 in HCB33 and MG1655 in TB-Kan
  • Transformation of Fusion-LuxP-pBAD33 constructs into DH5a

Wednesday, 09/24/2008

  • Glycerol-Stock of Fusion1-LuxP-pDK48 and Fusion2-LuxP-pDK48
  • O/N culture of Fusion-LuxP-pDK48 in HCB33 and MG1655 in LB
  • O/N culture of LuxS and GFP Plasmid from Marcus
  • Re-Sequencing of Fusion-1 and Fusion-2 
Correct sequence for both Fusion constructs confirmed

Thursday, 09/25/2008

  • Miniprep of Fusion-LuxP-pDK48 constructs, LuxS, GFP Plasmid from Marcus
  • Competent Cells (Top-10, UU1250)
  • Picked colonies of Fusion-LuxP-pBAD33

Friday, 09/26/2008

  • Miniprep of Fusion-LuxP-pBAD33 constructs and Digestion with HindIII (NEBuffer2)
    • 5 µl DNA
    • 1µl NEBuffer 2
    • 0.5µl HindIII
    • 3.5µl H2O
  • Expected Fragments
    • Fusion1-LuxP-pBAD33: 7616bp and 769bp
    • Fusion2-LuxP-pBAD33: 7631bp and 769bp
    • pBAD33 only: 5360bp
Double digestion of Fusion constructs in pBAD33 with BamHI/PstI and single digestion with HindIII. Double digestion yielded positive results for all constructs.
Double digestion of Fusion constructs in pBAD33 with BamHI/PstI and single digestion with HindIII. Double digestion yielded positive results for all constructs.
  • Double Transformations of Fusion-LuxP-pBAD33 and pDK4 into UU1250 (2 Amp-CM plates) as well as LuxS and pDK6 into DH5a (Amp-Kan plate(s))
  • Transformation of pDK4 into DH5a (CM plate)
  • Preparation of 2x 3mL LB-CM-Amp, 2x 5mL TB-CM-Amp, 1x 3mL LB-Amp-Kan, 1x 5mL TB-Amp-Kan, 1x 5mL LB-CM


Sunday, 09/28/2008

  • Picked colony of each Fusion-LuxP-pBad/pDK4 UU1250 cells and inoculated in 5 mL TB and 5 mL LB medium
  • No colonies on LuxS/pDK6 DH5a cell plate. 5 mL liquid culture of LuxS DH5a cells.
  • No colonies on pDK4 cell plate. Waiting until tomorrow


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