IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/29

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Raphaël Making Amp plates 40 plates Aleksandra and Théotime Miniprep stand.RBS /TetA(C) 1 and 2 pLux/RBS/GFP/term A and B (transformations from 07/27) Pmed/weakRBS/LuxR 1 and 2 Pstr/weakRBS/LuxR 1 and 2 Pmed /medRBS/LuxR 1 and 2 Pstr/medRBS/LuxR 1 and 2 Pmed/strRBS/LuxR 1 and 2 Pstr/strRBS/LuxR 1 and 2 stand.RBS/LacZ 1 and 2 weak RBS/LacZ 1 and 2 LuxI/term 1 and 2 attC term/attC/mRFP pLux/RBS/GFP/term 1 to 5 (transformations from 07/21) Théotime and Raphaël Restriction digest, incubation at 37°C for 2h. Restriction digests to do gel purification : 20µl stand.RBS /TetA(C) 1 + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x 20µl med.RBS /TetA(C) + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x 20µl weak.RBS /TetA(C) + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x 20µl stand.RBS /LacZ 1 + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x 20µl weak.RBS /LacZ 1 + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x 20µl LuxI/term 1 + 1µl EcoRI + 1µl XbaI + 2.5µl buffer4 10x + 0.25µl BSA 100x 20µl attC + 1µl SpeI + 1µl PstI + 2.5µl buffer2 10x + 0.25µl BSA 100x 20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x Restriction digests to verify the transformations : 5µl pLux/GFP 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl pLux/GFP 2 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl pLux/GFP 3 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl pLux/GFP 4 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl pLux/GFP A + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl pLux/GFP B + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl Pstr/weakRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl Pmed/weakRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl Pstr/medRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl Pmed /medRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl Pstr/strRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl Pmed/strRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water Aleksandra and Raphaël Gel electrophoresis (1.5% agarose, 100V) Since the yield of the gel purification was bad (between 5 and 30%), the experiment was done at the Necker's lab in order to control the role of materials and buffers. pLux/GFP 1 : 20µl + 5µl lb pLux/GFP 2 : 20µl + 5µl lb pLux/GFP 3 : 20µl + 5µl lb pLux/GFP 4 : 20µl + 5µl lb pLux/GFP 5 : 20µl + 5µl lb pLux/GFP A : 20µl + 5µl lb pLux/GFP B : 20µl + 5µl lb Pstr/weakRBS/LuxR 1 : 20µl + 5µl lb Pmed/weakRBS/LuxR 1 : 20µl + 5µl lb Pstr/medRBS/LuxR 1 : 20µl + 5µl lb Pmed /medRBS/LuxR 1 : 20µl + 5µl lb Ladder 1Kb (Fermentas) : 10 µL Pstr/strRBS/LuxR 1 : 20µl + 5µl lb Pmed/strRBS/LuxR 1 : 20µl + 5µl lb Ladder 1Kb (Fermentas) : 10 µL term/attC/mRFP : 25µl + 5µl lb - weak.RBS /TetA(C) 1 : 25µl + 5µl lb - med.RBS /TetA(C) 1 : 25µl + 5µl lb - stand.RBS /TetA(C) 1 : 25µl + 5µl lb - - weak.RBS /LacZ 1 : 25µl + 5µl lb - stand.RBS /LacZ 1  : 25µl + 5µl lb - Ladder 100bp : 10 µL -