IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/30

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Aleksandra Gel purification with the Quiagen kit Purification of the bands extracted yesterday : term/attC/mRFP weak.RBS /TetA(C) med.RBS /TetA(C) stand.RBS /TetA(C) weak.RBS /LacZ stand.RBS /LacZ Léa PCR purification with the Quiagen kit attC LuxI/term Léa, Aleksandra and Raphaël Nanodrop Quantification of DNA amounts in the minipreps from yesterday Sample name 260/280 260/230 ng/µl stand.RBS /TetA(C) 1 1.93 2.31 271.3 pLux/RBS/GFP/term A 1.90 2.18 244.8 Pmed/weakRBS/LuxR 1 1.91 1.52 87.8 Pstr/weakRBS/LuxR 1 1.94 2.28 88.4 Pmed /medRBS/LuxR 1 1.89 2.19 156.2 Pstr/medRBS/LuxR 1 1.91 2.04 83.9 Pmed/strRBS/LuxR 1 1.94 2.37 236.5 Pstr/strRBS/LuxR 1 1.96 2.43 101.3 stand.RBS/LacZ 1 1.88 1.68 224.7 weak RBS/LacZ 1 1.92 2.06 162.4 LuxI/term 1 1.91 2.18 325.2 attC 1.97 1.96 152.0 term/attC/mRFP 2.03 2.36 213.2 Quantification of DNA amounts in the gel purification and PCR purification samples Sample name 260/280 260/230 ng/µl term/attC/mRFP (gel purif.) 1.34 0.47 9.3 weak.RBS /TetA(C) (gel purif.) 1.67 0.64 27.0 med.RBS /TetA(C) (gel purif.) 1.90 0.54 24.2 stand.RBS /TetA(C) (gel purif.) 1.76 0.52 31.3 weak.RBS /LacZ (gel purif.) 1.45 0.47 11.8 stand.RBS /LacZ (gel purif.) 1.85 0.07 4.0 attC (PCR purif.) 1.42 0.70 19.1 LuxI/term (PCR purif.) 1.60 2.03 22.1 attC (PCR purif. from 07/23) 1.69 1.24 22.4 Aleksandra Ligation using around 150ng of the insert and 50µg of the vector. Incubation 1h at 25°C. 2µl attC (07/23) + 15µl term/attC/mRFP + 2µl buffer + 0.5µl ligase 2µl LuxI/term + 6µl weak.RBS /TetA(C) + 1µl buffer + 0.5µl ligase 2µl LuxI/term + 6µl med.RBS /TetA(C) + 1µl buffer + 0.5µl ligase 2µl LuxI/term + 6µl stand.RBS /TetA(C) + 1µl buffer + 0.5µl ligase 2µl LuxI/term + 15µl weak.RBS /LacZ + 2µl buffer + 0.5µl ligase 2µl LuxI/term + 30µl stand.RBS /LacZ + 3.6µl buffer + 0.5µl ligase Raphaël Transformation in TOP10 cells attC/term/attC/mRFP weak.RBS /TetA(C)/LuxI/term med.RBS /TetA(C)/LuxI/term stand.RBS /TetA(C)/LuxI/term weak.RBS /LacZ/LuxI/term stand.RBS /LacZ/LuxI/term pUC 19 positive control → there are colonies on every plate