IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/24

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Aleksandra and Raphaël


Gel extraction (from PCR products)

  • WTet-LuxI
  • MTet-LuxI
  • STet-LuxI


Restriction digest with XbaI and PstI : 15µl gel extraction products + 2x1µl enzyme + 2µl B2 + 0.2µl BSA

  • WTet-LuxI
  • MTet-LuxI
  • STet-LuxI


Restriction digest with XbaI, PstI and AlWNI : 8µl miniprep (13/08) + 3x0,5µl enzyme + 1,5µl B2 + 0.2µl BSA

  • WTet-LuxI
  • MTet-LuxI
  • STet-LuxI


Gel electrophoresis

  • WTet-LuxI (PCR product)
  • -
  • MTet-LuxI (PCR product)
  • -
  • STet-LuxI (PCR product)
  • -
  • WTet-LuxI (Triple restriction)
  • -
  • MTet-LuxI (Triple restriction)
  • -
  • STet-LuxI (Triple restriction)
  • Ladder 1Kb

Image:Gel2408 1.jpg


Gel extraction

  • WTet-LuxI (PCR product)
  • MTet-LuxI (PCR product)
  • STet-LuxI (PCR product)
  • WTet-LuxI (Triple restriction)
  • MTet-LuxI (Triple restriction)
  • STet-LuxI (Triple restriction)


Ligations

  • PmS-attC + WTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PmS-attC + WTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PmS-attC + MTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PmS-attC + MTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PmS-attC + STet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PmS-attC + STet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsS-attC + WTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsS-attC + WTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsS-attC + MTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsS-attC + MTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsS-attC + STet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsS-attC + STet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsM-attC + WTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsM-attC + WTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsM-attC + MTet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsM-attC + MTet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsM-attC + STet-LuxI (PCR) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • PsM-attC + STet-LuxI (Triple restr.) (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • pLux + LuxI (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)
  • WLac + LuxI (4µl + 10µl + 0.5µl ligase + 1,6µl buffer)


Controls :

  • LuxI (4µl + 0.5µl ligase + 1,6µl buffer + 10µl water)
  • PsM (4µl + 0.5µl ligase + 1,6µl buffer + 10µl water)
  • PsS (4µl + 0.5µl ligase + 1,6µl buffer + 10µl water)
  • PmS (4µl + 0.5µl ligase + 1,6µl buffer + 10µl water)


Transformations of DH5α with 5µL of ligation product, plating with 150µl

  • PmS-attC + WTet-LuxI (PCR)
  • PmS-attC + WTet-LuxI (Triple restr.)
  • PmS-attC + MTet-LuxI (PCR)
  • PmS-attC + MTet-LuxI (Triple restr.)
  • PmS-attC + STet-LuxI (PCR)
  • PmS-attC + STet-LuxI (Triple restr.)
  • PsS-attC + WTet-LuxI (PCR)
  • PsS-attC + WTet-LuxI (Triple restr.)
  • PsS-attC + MTet-LuxI (PCR)
  • PsS-attC + MTet-LuxI (Triple restr.)
  • PsS-attC + STet-LuxI (PCR)
  • PsS-attC + STet-LuxI (Triple restr.)
  • PsM-attC + WTet-LuxI (PCR)
  • PsM-attC + WTet-LuxI (Triple restr.)
  • PsM-attC + MTet-LuxI (PCR)
  • PsM-attC + MTet-LuxI (Triple restr.)
  • PsM-attC + STet-LuxI (PCR)
  • PsM-attC + STet-LuxI (Triple restr.)


Controls :

  • pLux + LuxI
  • WLac + LuxI
  • LuxI
  • PsM
  • PsS
  • PmS
  • pUC19 (positive control)


Overnight cultures

  • PsW-attC x2
  • PsM-attc x2
  • PsS-attC x2
  • PmS-attC x2
  • WLac
  • SLac
  • WTet-LuxI x2
  • MTet-LuxI x2
  • STet-LuxI x2
  • pLux
  • LuxI
  • I13507-pLux-GFP


Plating glycerols (verification)

  • WTet-LuxI
  • MTet-LuxI
  • STet-LuxI


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