IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/05

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Léa
Minipreps of 10 ml cultures, elution in 30µl warm water

  • attC/term/attC/mRFP, 11 minipreps from glycerol
  • RBS/LuxI/term, started from colony 1 from TURBOcell plate 1
  • RBS/LuxI/term, started from colony 1 from TURBOcell plate 2
  • RBS/LuxI/term, started from colony 2 from TURBOcell plate 2
  • RBS/LuxI/term, started from colony 1 from TOP10 plate
  • RBS/LuxI/term, started from colony 1 from TOP10 plate
  • term from glycerol


DNA quantification in the minipreps with the Nanodrop

Sample name 260/280 260/230ng/µl
attC/term/attC/mRFP 1.991.98 206.8
RBS/LuxI/term (TURBO1 col1)1.94 2.22 318.6
RBS/LuxI/term (TURBO2 col1) 1.95 2.27325.4
RBS/LuxI/term (TURBO2 col2) 1.95 2.31364.1
RBS/LuxI/term (TOP10 col1) 1.96 1.99246.5
RBS/LuxI/term (TOP10 col2) 1.932.17 292.4
term 1.982.20 299.9


Restriction digest

  • weakRBS/LacZα with XbaI and PstI : 20µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA
  • strRBS/LacZα with XbaI and PstI : 20µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA
  • weakRBS/TetA(C) with XbaI and PstI : 20µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA
  • medRBS/TetA(C) with XbaI and PstI : 20µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA
  • strRBS/tetA(C) with XbaI and PstI : 20µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA
  • pLux/GFP with XbaI and PstI : 20µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA
  • RBS/LuxI/term (TURBO1 col1) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzymes + 2.5µl B4 + 0.25µl BSA
  • RBS/LuxI/term (TURBO2 col1) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzymes + 2.5µl B4 + 0.25µl BSA
  • RBS/LuxI/term (TURBO2 col2) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzymes + 2.5µl B4 + 0.25µl BSA
  • RBS/LuxI/term (TOP10 col1) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzymes + 2.5µl B4 + 0.25µl BSA
  • RBS/LuxI/term (TOP10 col2) with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzymes + 2.5µl B4 + 0.25µl BSA
  • attC/term/attC/mRFP (miniprep) with XbaI and PstI : 100µl miniprep + 2x5µl enzymes + 12.5µl B2 + 1.3µl BSA
  • attC/term/attC/mRFP (miniprep) with SpeI and PstI : 10µl miniprep + 2x2µl enzymes + 5µl B2 + 0.5µl BSA


Raphaël
Restriction digest

  • attC/term/attC/mRFP (midiprep) with XbaI and PstI : 50µl miniprep + 2x5µl enzymes + 10.0µl B2 + 1µl BSA + 30µl water
  • Pmed with SpeI and PstI : 15µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA + 5µl water
  • Pstr with SpeI and PstI : 15µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA + 5µl water
  • Pmed/weakRBS/LuxR with SpeI and PstI : 15µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA + 5µl water
  • Pstr/weakRBS/LuxR with SpeI and PstI : 15µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA + 5µl water
  • Pmed/medRBS/LuxR with SpeI and PstI : 15µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA + 5µl water
  • Pstr/medRBS/LuxR with SpeI and PstI : 15µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA + 5µl water
  • Pmed/strRBS/LuxR with SpeI and PstI : 15µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA + 5µl water
  • Pstr/strRBS/LuxR with SpeI and PstI : 15µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA + 5µl water
  • term with EcoRI-HF and XbaI : 20µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA
  • LuxI with EcoRI-HF and SpeI : 20µl miniprep + 2x1µl enzymes + 2.5µl B2 + 0.25µl BSA


Gel electrophoresis, 1.5% agarose w/v, no EtB, 75V
Gel 1

  • attC/term/attC/mRFP (midiprep), 3 wells united
  • attC/term/attC/mRFP (miniprep cut with XbaI and PstI), 3 wells united
  • ladder 1kb
  • attC/term/attC/mRFP (miniprep cut with SpeI and PstI), 3 wells united
  • ladder 100bp
  • Pm
  • LuxI

Image:Gel0508 1.jpg Image:Gel0508 1cut.jpg


Gel 2

  • GFP
  • ladder 1kb
  • weakRBS/TetA(C)
  • -
  • medRBS/TetA(C)
  • -
  • standRBS/TetA(C)
  • -
  • weakRBS/LacZα
  • -
  • strRBS/LacZα
  • ladder 100bp

Image:Gel0508 2.jpg Image:Gel0508 2cut.jpg


Gel 3

  • RBS/LuxI/term (TURBO1 col1)
  • RBS/LuxI/term (TURBO2 col1)
  • RBS/LuxI/term (TURBO2 col2)
  • RBS/LuxI/term (TOP10 col1)
  • RBS/LuxI/term (TOP10 col2)
  • Ladder 100bp

Image:Gel0508 3.jpg Image:Gel0508 3cut.jpg


Aleksandra
Gel extraction

  • attC/term/attC/mRFP (midiprep)
  • attC/term/attC/mRFP (miniprep cut with XbaI and PstI)
  • mRFP
  • LuxI
  • Pm


Stéphane
PCR purification kit

  • Ps
  • Pm/weakRBS/LuxR
  • Ps/weakRBS/LuxR
  • Pm/medRBS/LuxR
  • Ps/medRBS/LuxR
  • Pm/standRBS/LuxR
  • Ps/standRBS/LuxR
  • term from 05/08
  • term from 04/08


Aleksandra
DNA quantifcation using Nanodrop

Sample name260/280 260/230 ng/µl
attC/term/attC/mRFP (midiprep) 2.770.02 3.8
attC/term/attC/mRFP (miniprep cut with XbaI and PstI) 2.46 0.0410.8
mRFP 2.53 0.05 7.8
LuxI 4.920.01 2.2
Pm 1.81 0.089.5
Ps 1.81 0.6711.0
Pm/weakRBS/LuxR 1.62 1.07 8.6
Ps/weakRBS/LuxR1.54 0.52 5.0
Pm/medRBS/LuxR 1.710.97 6.9
Ps/medRBS/LuxR 2.07 0.806.6
Pm/standRBS/LuxR 1.71 0.63 4.2
Ps/standRBS/LuxR 1.84 1.8023.4
term from 05/08 1.76 0.632.8
term from 04/08 2.121.68 13.5


Ligation

  • 5µl Pmed + 15µl mRFP + 2.7µl buffer + 0.5µl ligase
  • 5µl Pst + 15µl mRFP + 2.7µl buffer + 0.5µl ligase
  • Pmed/strRBS/LuxR + attC/term/attC/mRFP
  • Pmed/medRBS/LuxR + attC/term/attC/mRFP
  • Pmed/weakRBS/LuxR + attC/term/attC/mRFP
  • Pstr/weakRBS/LuxR + attC/term/attC/mRFP
  • Pstr/medRBS/LuxR + attC/term/attC/mRFP
  • Pstr/strRBS/LuxR + attC/term/attC/mRFP
  • Ps ctrl
  • Pm ctrl
  • Pm/weakRBS/LuxR ctrl
  • Pstr/strRBS/LuxR ctrl


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