IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/10

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Raphaël and Théotime Minipreps, elution with 35µl warm water : 11 attC/term/attC/mRFP Pst/weakRBS/LuxR Pst/medRBS/LuxR Pmed/weakRBS/LuxR Pmed/medRBS/LuxR Pmed/stRBS/LuxR Pmed Pst wRBS/TetA(C) medRBS/TetA(C) Nanodrop Sample name 260/280 260/230 ng/µl attC/term/attC/mRFP 2.08 1.88 119.5 Pmed/wRBS/LuxR 2.00 1.98 162.0 Ps/wRBS/LuxR 1.75 1.67 140.4 Pm/mRBS/luxR 1.97 1.94 265.9 Ps/mRFP/LuxR 2.06 1.79 117.6 Pm/sRBS/LuxR 1.97 1.88 188.4 Restriction digest of today's minipreps Pst/weakRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes Pst/medRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes Pmed/weakRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes Pmed/medRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes Pmed/stRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes wRBS/TetA(C) with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes medRBS/TetA(C) with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes Restriction digest of previuos minipreps: Pst/stRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes stRBS/TetA(C) with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes stLacZα with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes K081014 col2 with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes I13207 col2 with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes K081014 with EcoRI-HF and XbaI : SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes pLux/RBS with EcoRI-HF and XbaI : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes Gel electrophoresis Gel 1 attC/term/attC/mRFP in a big well - weakRBS/TetA(C) - medRBS/TetA(C) - stRBS/TetA(C) Ladder 1kb Gel 2 stRBS/LacZα Ladder 100bp K081014 - I13507 Ladder 1kb Aleksandra Gel extraction Elution in 35µl warm water attC/term/attC/mRFP weakRBS/TetA(C) medRBS/TetA(C) stRBS/TetA(C) stRBS/LacZα K081014 I13507 Raphaël PCR purification kit Pm/wRBS/LuxR Ps/wRBS/LuxR Pm/mRBS/luxR Ps/mRBS/LuxR Pm/sRBS/LuxR Ps/sRBS/LuxR LuxI pLux/GFP Nanodrop Sample name 260/280 260/230 ng/µl mRBS/TetA(C) 1.70 0.07 6.6 wRBS/tetA(C) 1.41 0.07 5.1 sRBS/TetA(C) 2.01 0.07 7.3 sRBS/LacZα 1.59 0.05 3.0 K081014 2.31 0.11 7.4 I13507 2.04 0.08 7.5 attC/term/attC/mRFP 1.58 0.07 12.8 pLux/GFP 1.80 1.51 45.8 Pm/wRBS/LuxR 1.71 1.21 31.5 Ps/wRBS/LuxR 1.97 0.68 8.7 Pm/mRBS/luxR 1.81 1.46 44.4 Ps/mRBS/LuxR 1.42 0.51 15.9 Pm/sRBS/LuxR 1.77 0.94 25.8 Ps/sRBS/LuxR 1.37 0.39 5.2 Ps 1.58 0.64 26.6 Ligation mRBS/TetA(C)+RBS/LuxI : 10µl + 1µl + 1.2µl buffer + 0.5µl ligase wRBS/TetA(C)+RBS/LuxI : 10µl + 1µl + 1.2µl buffer + 0.5µl ligase stRBS/TetA(C)+RBS/LuxI : 10µl + 1µl + 1.2µl buffer + 0.5µl ligase stRBS/LacZα+RBS/LuxI : 10µl + 1µl + 1.2µl buffer + 0.5µl ligase RBS/mRFP/t(K081014)+pLux/GFP : 5µl + 1µl(1/4dilution) + 0.7µl buffer + 0.5µl ligase RBS/mRFP/tt(I13507)+pLux/GFP : 5µl + 1µl(1/4dilution) + 0.7µl buffer + 0.5µl ligase Pm/wRBS/LuxR +attC/term/attC/mRFP : 1µl (1/3dilutuion) + 3µl + 1µl buffer + 0.5µl ligase + 4µl water Ps/wRBS/LuxR +attC/term/attC/mRFP : 2µl + 3µl + 1µl buffer + 0.5µl ligase + 4µl water Pm/mRBS/luxR +attC/term/attC/mRFP : 1µl (1/4dilutuion) + 3µl + 1µl buffer + 0.5µl ligase + 4µl water Ps/mRBS/LuxR +attC/term/attC/mRFP : 1µl + 3µl + 1µl buffer + 0.5µl ligase + 4µl water Pm/sRBS/LuxR +attC/term/attC/mRFP : 1µl (1/2dilutuion) + 3µl + 1µl buffer + 0.5µl ligase + 4µl water Ps/sRBS/LuxR +attC/term/attC/mRFP : 2µl + 3µl + 1µl buffer + 0.5µl ligase + 4µl water Controls: RBS/LuxI : 10µl water + 1µl + 1.2µl buffer + 0.5µl ligase pLux/GFP : 5µl water + 1µl(1/4dilution) + 0.7µl buffer + 0.5µl ligase Pm/wRBS/LuxR : 1µl (1/3dilutuion) + 1µl buffer + 0.5µl ligase + 7µl water Ps/wRBS/LuxR : 2µl + 1µl buffer + 0.5µl ligase + 7µl water Pm/mRBS/luxR : 1µl (1/4dilutuion)+ 1µl buffer + 0.5µl ligase + 7µl water Ps/mRBS/LuxR : 1µl + 1µl buffer + 0.5µl ligase + 7µl water Pm/sRBS/LuxR : 1µl (1/2dilutuion) + 1µl buffer + 0.5µl ligase + 7µl water Ps/sRBS/LuxR : 2µl + 1µl buffer + 0.5µl ligase + 7µl water Aleksandra Overnight cultures in 10ml cultures Pst/weakRBS/LuxR Pst/sRBS/LuxR Pmed wRBS/TetA(C) medRBS/TetA(C) sRBS/TetA(C) wRBS/LacZα sRBS/LacZα Plating As some of our overnight cultures had no bvacteria, we decided to plate the glycerols to verify if they work and do some new glycerols. Ps/sRBS/LuxR wRBS/LacZα stRBS/LacZα stRBS/TetA(C)