IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/03

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Théotime and Raphaël Transformation of TURBO cells, incubation at 37°C around 10h RBS/LuxI/term (ligation of 26/07), with 50µl RBS/LuxI/term (ligation of 26/07), with 100µl positive control Aleksandra Minipreps of 10ml, elution in 35µl warm water attC (from glycerol stock) term/attC/mRFP (from glycerol stock) attC/term/attC/mRFP #10 (from yesterday's glycerol stock) attC/term/attC/mRFP #11 (from yesterday's glycerol stock) DNA quantification in the minipreps using the nanodrop Sample name 260/280 260/230 ng/µl attC 2.13 1.69 135.2 term 2.13 2.07 169.9 attC/term/attC/mRFP #10 2.02 1.91 118.0 Léa and Aleksandra Digestion attC (today's miniprep) with SpeI+PstI (B2) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA term/attC/mRFP (today's miniprep) with XbaI+PstI (B3) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA 16 minipreps attC/term/attC/mRFP (yesterday's and today's minipreps) with EcoRI-HF + SpeI (B4) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA RBS/LuxI/term (miniprep from 26/07) with EcoRI-HF + SpeI (B4) : 10µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA + 6µl water RBS/LuxI (miniprep from 24/07) with EcoRI-HF + SpeI (B4) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA term (miniprep from 24/07) with EcoRI-HF + SpeI (B4) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA Gel electrophoresis, 15%agarose, no EtB Gel 1, 75V attC/term/attC #1 - attC/term/attC #2 ladder 100bp attC/term/attC #3 - attC/term/attC #4 - attC/term/attC #5 - attC/term/attC #6 - attC/term/attC #7 - attC/term/attC #8 - attC/term/attC #9 Gel 2, 75V attC/term/attC #10 - attC/term/attC #11 - - attC/term/attC #12 - attC/term/attC #13 - attC/term/attC #14 ladder 1kb attC/term/attC #15 #10 and #11 have worked! Gel 3, 50V attC/term/attC #16 attC - term/attC/mRFP - RBS/LuxI/term - RBS/luxI - ladder 100bp - ladder 1kb Gel extraction, elution in 30µl warm water RBS/LuxI attC/term/attC #10 attC/term/attC #11 PCR kit purification term DNA quantification after gel extraction and PCR purification kit using the nanodrop Sample name 260/280 260/230 ng/µl RBS/LuxI attC/term/attC #10 attC/term/attC #11 term Léa Ligation 25µl LuxI miniprep + 2µl term miniprep + 1µl ligase + 3µl buffer 2µl term miniprep + 1µl ligase + 1µl buffer + 6µl water (control) Léa, Raphaël and Aleksandra Transformation of TOP10 cells, incubation at 37°C overnight 25µl LuxI miniprep 2µl term miniprep Raphaël and Aleksandra Overnight cultures, 10ml with Amp Pm Ps Pm/weakRBS/LuxR Ps/weakRBS/LuxR Pm/medRBS/LuxR Ps/medRBS/LuxR Pm/standRBS/LuxR Ps/standRBS/LuxR RBS/LuxI term Overnight cultures, 5x20ml with Kan attC/term/attC/mRFP #10 Aleksandra Overnight cultures started from this morning's TURBO cell plates, 2 colonies per plate RBS/LuxI/term