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 File: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