IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/13

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Raphaël Restriction digest 2h, 37°C (10µL of minipreped DNA) BBa_J23119 cut by SpeI + PstI BBa_J23110 cut by SpeI + PstI BBa_J37033 cut by XbaI + PstI BBa_R0062 cut by SpeI + PstI BBa_E0240 cut by XbaI + PstI Raphaël and Léa DNA Gel electrophoresis (agarose 1,0% w/v) (gel extraction for purification of DNA) After migration, we made a bath with EtBr during 5-10 min. Protocol (6 wells) 10 µL of ladder 1Kb 17 µL of digested DNA (J23119 - SpeI+PstI) + 3 µL of loading buffer 6x 17 µL of digested DNA (J23110 - SpeI+PstI) + 3 µL of loading buffer 6x 17 µL of digested DNA (R0062 - SpeI+PstI) + 3 µL of loading buffer 6x 17 µL of digested DNA (E0240 - XbaI+PstI) + 3 µL of loading buffer 6x 17 µL of digested DNA (J37033 - XbaI+PstI) + 3 µL of loading buffer 6x Migration : 50V, 1h30 double digestion of J37033 (LuxR) failed. Gel extraction - Kit Qiagen Ligation (T4 ligase), 1h at room temperature vector : R0062 (Plux) cut by SpeI + PstI insert : E0240 (RBS+GFP+ter) cut by XbaI + PstI Transformation Transformation into TOP10 - pSB AmpR with biobricks - Plating 100µL of each on the plates (Amp) - Incubation overnight : Product of ligation (R0062+E0240) -> Transformation didn't work Restriction digest 1h, 37°C (10µL of minipreped DNA) Again because the double digestion of J37033 didn't work and we have forgotten to digest 2 other biobricks BBa_J23119 cut by SpeI + PstI BBa_J23110 cut by SpeI + PstI BBa_J37033 cut by XbaI + PstI BBa_R0062 cut by SpeI + PstI BBa_E0240 cut by XbaI + PstI BBa_K081008 cut by SpeI + EcoRI BBa_B0015 cut by EcoRI + XbaI DNA Gel electrophoresis (agarose 1,0% w/v) (gel extraction for purification of DNA) After migration, we made a bath with EtBr during 5-10 min. Protocol (6 wells) 10 µL of ladder 1Kb 17 µL of digested DNA (J37033 - XbaI+PstI) + 3 µL of loading buffer 6x 17 µL of digested DNA (B0015 - EcoRI+XbaI) + 3 µL of loading buffer 6x 17 µL of digested DNA (K081008 - SpeI+EcoRI) + 3 µL of loading buffer 6x Migration : 50V, 1h double digestion of J37033 (LuxR) failed again ! Gel extraction - Kit Qiagen Ligation (T4 ligase), overnight in cold water vector : K081008 (LuxI) cut by SpeI + EcoRI insert : B0015 (ter) cut by EcoRI + XbaI Aleksandra Transformation Transformation into TOP10 - Plating 100µL of each on the plates (Amp) - Incubation overnight : Product of ligation (attC+ter+attC+mRFP) -> Transformation didn't work, the product of ligation is Kanamycin resistant NOT Ampicillin resistant !!! DNA Gel electrophoresis (agarose 1,0% w/v) (control of the ligation) 2 µL of EtBr was added in the gel Protocol (6 wells) 3 µL of ladder 1Kb + T4 ligase (incubation 1h at room temperature) 3 µL of ligation control 1 (vector only) + 3 µL of loading buffer 2x 3 µL of ligation control 2 (vector + ligase) + 3 µL of loading buffer 2x 7 µL of ladder 1Kb 3 µL of ligation product (1 µL) non digested + 3 µL of loading buffer 2x 3 µL of ligation product (1 µL) non digested + 3 µL of loading buffer 2x Migration : 50V, 30 min