IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/28

Memo-Cell	Main project page Previous entry      Next entry
Antoine Minipreps Vf=35 uL B0015 115.4 ng/uL B0015 200.4 ng/uL S200 174 ng/uL Restriction digest 2h 37°C follow by inactivation at 80°C 20 min Vf=20 uL Sample name DNA (1ug) enzyme Buffer H2O BSA B0015 (200.4 ng/uL) 5 1 X/P 2(B3) 10.8 0.2 B0015 5 1 X/P 2(B3) 10.8 0.2 B0015 5 1 X/P 2(B3) 10.8 0.2 B0015 5 1 X/P 2(B3) 10.8 0.2 S200 5.75 1 S/P 2 (B2) 10.05 0.2 S200 5.75 1 S/P 2 (B2) 10.05 0.2 Electrophoresis Gel purification Ligation follow by inactivation 65°C 10 min Vf=10uL Vecteur (10ng) Insert(30ng) Buffer ligase H2O T4 ligase P1003(cut S+P) 0.7 BOO15(cut X+P) 2.1 1 2(B3) 5.7 0.5 C0012(cut S+P) 0.9 BOO15(cut X+P) 2.1 1 2(B3) 5.5 0.5 S173(cut S+P) 0.6 BOO15(cut X+P) 2.1 1 2(B3) 5.8 0.5 S200(cut S+P) 1.5 BOO15(cut X+P) 2.1 1 2(B3) 4.9 0.5 Transformation
Population counter	Main project page Previous entry      Next entry
Raphaël Ethanol precipitation of the term/attC/mRFP (gel purification) to concentrate the sample. Following this protocol : [1] Elution with 20µL of water Aleksandra Miniprep 10 mL of culture, following Eric's advices : skip the step with PB buffer, thoroughly wipe the tube and the column after wash with PE buffer, elution with 50µL of water at 55°C. pSulib attC pSulib term/attC/mRFP TOP10 pSB1A2 pLux (R0062) TOP10 pSB1A2 RBS/GFP/term (E0240) TOP10 pSB1A2 pLux/GFP (1rst colony on the plate) TOP10 pSB1A2 pLux/GFP (2nd colony on the plate) TOP10 pSB1A2 pLux/GFP (3rd colony on the plate) TOP10 pSB1A2 pLux/GFP (4th colony on the plate) Nanodrop quantification of DNA amounts in our samples Sample name 260/280 260/230 ng/µl attC 2.00 1.89 142.0 term/attC/mRFP 1.98 2.14 163.8 pLux 1.94 2.32 217.5 GFP 1.92 2.37 274.1 pLux/GFP 1 1.93 2.30 208.7 pLux/GFP 2 1.94 2.23 260.6 pLux/GFP 3 1.95 2.26 264.3 pLux/GFP 4 1.91 2.31 326.2 term/attC/mRFP (ethanol prec.) 1.27 0.12 5.2 Aleksandra and Raphaël Restriction digest, incubation at 37°C for 2h. 20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x 20µl RBS/GFP/term + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x 20µl pLux + 1µl SpeI + 1µl PstI + 2.5µl buffer4 10x + 0.25µl BSA 100x 20µl attC + 1µl SpeI + 1µl PstI + 2.5µl buffer4 10x + 0.25µl BSA 100x 5µl pLux/GFP 1 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl pLux/GFP 2 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl pLux/GFP 3 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water 5µl pLux/GFP 4 + 1µl XbaI + 1µl SpeI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water Aleksandra and Théotime Gel electrophoresis (1.5% agarose, 50V) attC : 25µl + 5µl lb Ladder 1Kb term/attC/mRFP : 25µl + 5µl lb - pLux : 25µl + 5µl lb - GFP : 25µl + 5µl lb - pLux/GFP 1 : 20µl + 5µl lb pLux/GFP 2 : 20µl + 5µl lb pLux/GFP 3 : 20µl + 5µl lb pLux/GFP 4 : 20µl + 5µl lb Gel purification with the Quiagen kit, elution in 50µl warm water. Nanodrop quantification of DNA amounts in our samples Sample name 260/280 260/230 ng/µl attC (gel purif.) 2.67 0.03 5.0* term/attC/mRFP (gel purif.) 2.53 0.05 3.9* pLux (gel purif.) 2.18 0.15 11.0 GFP (gel purif.) 1.84 0.25 35.0 *These samples have no DNA : no peak at 260nm on the Nanodrop. The yields of DNA are following : 8.8% for attC, 6% for term/attC/mRFP, 13% for pLux, 32% for RBS/GFP/term Ligation 50ng of the vector and 150ng of insert in about 10μL of total volume. Ligation at 16°C overnight. 15μL GFP (E0240) + 1.5μL pLux (R0062) + 2μL buffer 10x + 0.5μL T4 ligase + 1µl water Overnight culture of the transformations from 07/27 (colonies on all the plates) 10mL LB + Amp, 2 colonies for each transformation TetA(C) + stand.RBS RBS/GFP/term + pLux weakRBS/LuxR + Pmed weakRBS/LuxR + Pstr medRBS/LuxR + Pmed medRBS/LuxR + Pstr strRBS/LuxR + Pmed strRBS/LuxR + Pstr LacZ + weak RBS LacZ + stand RBS LuxI + term Overnight culture from the glycerol stock 10mL LB 2x attC 2x term/attC/mRFP Overnight culture of the transformation from 07/21, 5 colonies pLux/GFP/term