IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/14

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Aleksandra Minipreps from 10ml culture, elution in 35µl warm water attP HK wt col1 attP HK wt col2 attP HK mut col1 attP HK mut col2 attP λ mut col1 attP λ mut col2 Nanodrop of the minipreps Sample name 260/280 260/230 ng/µl attP HK wt col1 1.96 2.35 340.1 attP HK wt col2 1.92 2.32 411.5 attP HK mut col1 1,92 2.29 329.1 attP HK mut col2 1.97 2.30 282.7 attP λ mut col1 1.95 2.24 282.7 attP λ mut col2 1.93 2.21 264.0
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Aleksandra Gel extraction, elution in 35µl warm water Ps/sRBS/LuxR/attC/term/attC Pm/sRBS/LuxR/attC/term/attc Ps/mRBS/LuxR/attC/term/attc Ps/wRBS/LuxR/attC/term/attc RBS/LuxI PCR kit purification, elution in 35µl warm water wRBS/LacZα stRBS/lacZα pLux Pm/wRBS/LuxR Pm/mRBS/LuxR Nanodrop Sample name 260/280 260/230 ng/µl Ps/sRBS/LuxR/attC/term/attC 1.86 0.18 63.2 Pm/sRBS/LuxR/attC/term/attc 1.82 0.36 75.2 Ps/mRBS/LuxR/attC/term/attc 1.74 0.43 27.8 Ps/wRBS/LuxR/attC/term/attc 1.83 0.42 33.3 RBS/LuxI 1.67 0.03 8.2 wRBS/LacZα 1.83 1.99 28.3 stRBS/lacZα 1.77 1.18 42.7 pLux 1.76 1.51 56.1 Pm/wRBS/LuxR 1.56 0.76 42.5 Pm/mRBS/LuxR 1.75 1.76 35.5 Ligation for 45mins at 25°C pLux+LuxI : 1µl + 18µl + 2.2 buffer + 0.5µl ligase Pm/wRBS+attC/t/attC/mRFP : 0.5µl + 5µl + 0.5µl boffer + 0.5µl ligase Pm/mRBS+attC/t/attC/mRFP : 0.5µl + 5µl + 0.5µl boffer + 0.5µl ligase Controls : pLux : 1µl + 18µl water + 2.2 buffer + 0.5µl ligase Pm/wRBS : 0.5µl + 5µl water + 0.5µl boffer + 0.5µl ligase Pm/mRBS : 0.5µl + 5µl water + 0.5µl boffer + 0.5µl ligase Transformation of DH5α cells prepares by last year's iGEM team (P1) With today's ligations: pLux+LuxI Pm/wRBS+attC/t/attC/mRFP Pm/mRBS+attC/t/attC/mRFP Controls : pLux Pm/wRBS Pm/mRBS positive control (pUC19) Double transformation to verify the phenotype (RFP) when the integrase is activated pSUlib (pBAD/IntI, Kan) and pSB1A2 (Ps/sRBS/attC/term/attC/mRFP, Amp) pSUlib (pBAD/IntI, Kan) and pSB1A2 (Ps/mRBS/attC/term/attC/mRFP, Amp) pSUlib (pBAD/IntI, Kan) and pSB1A2 (Ps/wRBS/attC/term/attC/mRFP, Amp) pSUlib (pBAD/IntI, Kan) and pSB1A2 (Pm/sRBS/attC/term/attC/mRFP, Amp) Overnight cultures wRBS/TetA(C) mRBS/TetA(C) stRBS/TetA(C) wRBS/LacZα sRBS/LacZα RBS/LuxI