IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/01

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Aleksandra Glycerols of the yesterday's overnight cultures 15%glycerol (375µl 60%glycerol + 1125µl cultute), 2 for each (different colonies): attC/term/attC/mRFP weak RBS/TetA(C)/LuxI/term medium RBS/TetA(C)/LuxI/term strong RBS/TetA(C)/LuxI/term weak RBS/LacZ alpha/LuxI/term strong RBS/LacZ alpha/LuxI/term Minipreps of the yesterday's overnight cultures, 2 for each part(cultures starting from colonies #1 and #2 on each plate) Elution in 35µl EB buffer attC/term/attC/mRFP weak RBS/TetA(C)/LuxI/term medium RBS/TetA(C)/LuxI/term strong RBS/TetA(C)/LuxI/term weak RBS/LacZ alpha/LuxI/term strong RBS/LacZ alpha/LuxI/term Quantification of the DNA in the minipreps by Nanodrop Sample name 260/280 260/230 ng/µl attC/term/attC/mRFP 1 1.97 2.11 159.6 attC/term/attC/mRFP 2 1.96 1.87 195.3 weakRBS/TetA(C)/LuxI/term 1 1.96 2.28 381.3 weakRBS/TetA(C)/LuxI/term 2 1.96 2.30 291.9 mediumRBS/TetA(C)/LuxI/term 1 1.96 2.26 353.2 mediumRBS/TetA(C)/LuxI/term 2 1.95 2.28 490.1 standardRBS/TetA(C)/LuxI/term 1 1.96 2.27 425.2 standardRBS/TetA(C)/LuxI/term 2 1.96 2.31 362.6 weakRBS/LacZ alpha/LuxI/term 1 1.96 2.20 260.2 weakRBS/LacZ alpha/LuxI/term 2 1.97 2.28 273.3 standardRBS/LacZ alpha/LuxI/term 1 1.98 2.24 248.6 standardRBS/LacZ alpha/LuxI/term 2 1.97 2.23 265.0 Restriction digests Preparative digests (20µl miniprep + 2x1µl enzymes + 2.5µl buffer4 + 0.25µl BSA) Minipreps from today (only with minipreps from colony #1): attC/term/attC/mRFP with EcoRI-HF+XbaI weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI medium RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI strong RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI strong RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI Minipreps from 29/07: pLux/GFP with EcoRI-HF+XbaI Ps/weakRBS/LuxR with EcoRI-HF+SpeI Pm/weakRBS/LuxR with EcoRI-HF+SpeI Ps/mediumRBS/LuxR with EcoRI-HF+SpeI Pm/mediumRBS/LuxR with EcoRI-HF+SpeI Ps/stongRBS/LuxR with EcoRI-HF+SpeI Pm/strongRBS/LuxR with EcoRI-HF+SpeI Verification digests (5µl miniprep + 12µl water + 2x1µl enzymes + 2µl buffer4 + 0.2µl BSA) attC/term/attC/mRFP (miniprep from colony #1) with EcoRI-HF+PstI attC/term/attC/mRFP (miniprep from colony #2) with EcoRI-HF+PstI Gel electrophoresis (1.5%agarose w/v, 50V) 20µl restriction digest+5µl 6x loading buffer weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI weak RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI ladder medium RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI - strong RBS/TetA(C)/LuxI/term with EcoRI-HF+SpeI - weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI - strong RBS/LacZ alpha/LuxI/term with EcoRI-HF+SpeI - attC/term/attC/mRFP #1 (digested with EcoRI-HF+PstI for verification) attC/term/attC/mRFP #2 (digested with EcoRI-HF+PstI for verification) Ps/weakRBS/LuxR with EcoRI-HF+SpeI - Pm/weakRBS/LuxR with EcoRI-HF+SpeI - Ps/mediumRBS/LuxR with EcoRI-HF+SpeI - Pm/mediumRBS/LuxR with EcoRI-HF+SpeI - Ps/stongRBS/LuxR with EcoRI-HF+SpeI - Pm/strongRBS/LuxR with EcoRI-HF+SpeI ladder Gel extraction Elution in 35µl warm water weak RBS/TetA(C)/LuxI/term medium RBS/TetA(C)/LuxI/term strong RBS/TetA(C)/LuxI/term weak RBS/LacZ alpha/LuxI/term strong RBS/LacZ alpha/LuxI/term Ps/weakRBS/LuxR Pm/weakRBS/LuxR Ps/mediumRBS/LuxR Pm/mediumRBS/LuxR Pm/strongRBS/LuxR DNA quantification after gel extraction. Measured with the Nanodrop Sample name 260/280 260/230 ng/µl weakRBS/TetA(C)/LuxI/term 1 1.77 0.13 27.1 mediumRBS/TetA(C)/LuxI/term 1 1.68 0.10 23.2 standardRBS/TetA(C)/LuxI/term 1 1.77 0.16 27.3 weakRBS/LacZ alpha/LuxI/term 1 1.77 0.02 8.1 standardRBS/LacZ alpha/LuxI/term 1 1.59 0.03 9.3 Pm/standRBS/LuxR 1.90 0.01 3.5* Pm/medRBS/LuxR 1.22 0.02 1.5* Ps/medRBS/LuxR 2.20 0.03 2.5 Pm/weakRBS/LuxR 1.06 0.01 2.3* Ps/weakRBS/LuxR 1.55 0.06 7.6 *Apparently no DNA in the sample : no peak on the Nanodrop. PCR purification kit pLux/GFP attC/term/attC/mRFP (digested with EcoRI-HF and Xba-I for future ligation) DNA quantification after PCR purification kit. Measured with the Nanodrop Sample name 260/280 260/230 ng/µl attC/term/attC/mRFP (from colony#1) 1.52 0.59 8.8 attC/term/attC/mRFP (from colony#2) 1.69 0.08 9.8 pLux/GFP (A from 29/07) 1.69 1.36 77.3 Ligation at 16°C overnight 1µl pLux/GFP + 6µl stand.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase 1µl pLux/GFP + 6µl med.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase 1µl pLux/GFP + 6µl weak.RBS/TetA(C)/LuxI/term + 1µl buffer + 1.5µl water + 0.5µl ligase 1µl pLux/GFP + 16.5µl stand.RBS/LacZα/LuxI/term + 2µl buffer + 0.5µl ligase 1µl pLux/GFP + 16.5µl weakRBS/LacZα/LuxI/term + 2µl buffer + 0.5µl ligase control 1 : 1µl pLux/GFP + 1µl buffer + 7.5µl water + 0.5µl ligase control 2 : 6µl stand.RBS/TetA(C)/LuxI/term + 1µl buffer + 2.5µl water + 0.5µl ligase Overnight cultures 12ml cultures LB+Kan started from 16 different colonies on the attC/term/attC/mRFP transformation plates from yesterday 10ml culture LB+Kan of attC (from glycerol) 10ml culture LB+Kan of term/attC/mRFP (from glycerol)