Team:Heidelberg/Notebook/Sensing Group/Notebook/9thweek

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Contents

Tuesday, 09/30/2008

  • Miniprep of LuxS+pDK6 and F1/F2-Luxp-pBAD33
  • Digestion of GeneArt Fusion constructs with EcoRI/PstI (NEBuffer EcoRI + BSA) and Gel-Extraction
  • Digestion of GeneArt Fusion constructs with EcoRI/PstI (NEBuffer EcoRI + BSA) and Gel-Extraction

Wednesday, 10/01/2008

  • Miniprep of Fusion-LuxP-pBAD33 (both constructs)
  • Digestion of pSB1A3 with EcoRI,PstI,NdeI (3µl DNA, 40 µl total volume)
  • Digestion of pSB2K3 (10µl DNA, 20µl total volume) with EcoRI/PstI (NEBuffer EcoRI + BSA)
PCR GeneArt Fusion constructs with Prefix/Suffix primer yielded no product. F1/F2 with prefix/speI primer were positive, but mutation strategy dropped later. A mix of LuxS and pDK6 was added on the gel to check if double transformation was successfull. Digestions of pSB1A3 and pSB2K3 with EcoRI/Pst were supposed to be used for subsequent standardization of parts

Thursday, 10/02/2008

  • PCR like yesterday overnight, but with 42°C annealing and as template with EcoRI/PstI digested Fusion constructs
  • Maxiprep of GeneArt Fusion Constructs and pSB2K3
  • PCR Purification of F1/F2 Prefix/SpeI PCR products
  • Digestion of F1-prefix/SpeI and F2-prefix/SpeI with EcoRI/SpeI (NEBuffer EcoRI + BSA,610 bp band)
  • Digestion of F1/F2-LuxP-pDK48 with EcoRI,NotI,XbaI
    • 4686 bp
    • 1330 bp (wanted band)
    • 599 bp
    • 587 bp
  • Mutagenesis PCR of LuxS (LuxSmutF/LuxSmutR) and LuxP (LuxPmutF/LuxPmutR) overnight

Friday, 10/03/2008

  • Digestion of mutagenesis PCR products with DpnI, 1h @ 37 °C

--> Analysis on 1% Agarose-Gel --> No band visible

  • Ligation of F1/2 (GA) with pSB1A3 and pSB2K3, 1h @ RT

--> Transformation into DH5a. Plated over weekend.

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