IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/02

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Léa and Théotime
Glycerols with 15%glycerol (375µl of 60% glycerol + 1125µl culture), all made in double for -20°C and -80°C:

  • 16 cultures from the attC/term/attC/mRFP transformation plate from 31/07


Minipreps
Elution in 35µl of warm water

  • attC
  • term/attC/mRFP
  • 16 cultures from the attC/term/attC/mRFP transformation plate from 31/07


Restriction digests
Preparative digests (20µl miniprep + 2x1µl enzymes + 2.5µl buffer + 0.25µl BSA)

  • attC with SpeI+PstI (buffer2)
  • term/attC/mRFP with XbaI+PstI (buffer3)


Verification digests (10µl miniprep + 6µl water + 2x1µl enzymes + 2µl buffer4 + 0.2µl BSA)

  • attC/term/attC/mRFP (miniprep from colony #1 to #16) with EcoRI-HF+PstI


Gel electrophoresis (1.5%agarose w/v, 50V)
20µl restriction digest + 5µl 5x loading buffer

  • ladder 1Kb
  • attC/term/attC/mRFP #1 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #2 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #3 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #4 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #5 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #6 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #7 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #8 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #9 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #10 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #11 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #12 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #13 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #14 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #15 (digested with EcoRI-HF+PstI for verification)
  • attC/term/attC/mRFP #16 (digested with EcoRI-HF+PstI for verification)

Gel0208 1.jpg
Unfortunately, the ladder was added in every well instead of the loading buffer...


  • term/attC/mRFP with XbaI+PstI
  • -
  • attC with SpeI+PstI
  • -
  • -
  • ladder 1Kb

Gel0208 2.jpg


Aleksandra


Restriction digests
Verification digests (5µl miniprep + 11µl water + 2x1µl enzymes + 2µl buffer2 + 0.2µl BSA)

  • weak RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
  • medium RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
  • standard RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
  • weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+PstI
  • standard RBS/LacZ alpha/LuxI/term with EcoRI-HF+PstI
  • weak RBS/TetA(C) with EcoRI-HF+PstI
  • medium RBS/TetA(C) with EcoRI-HF+PstI
  • standard RBS/TetA(C) with EcoRI-HF+PstI
  • weak RBS/LacZ alpha with EcoRI-HF+PstI
  • standard RBS/LacZ alpha with EcoRI-HF+PstI
  • LuxI/term with EcoRI-HF+PstI


Gel electrophoresis (1.5%agarose w/v, 50V)
20µl restriction digest + 5µl 5x loading buffer

  • -
  • weak RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
  • weak RBS/LacZ alpha with EcoRI-HF+PstI
  • medium RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
  • medium RBS/TetA(C) with EcoRI-HF+PstI
  • standard RBS/TetA(C)/LuxI/term with EcoRI-HF+PstI
  • standard RBS/TetA(C) with EcoRI-HF+PstI
  • ladder 1Kb
  • ladder 100bp
  • LuxI/term with EcoRI-HF+PstI
  • -
  • weak RBS/LacZ alpha/LuxI/term with EcoRI-HF+PstI
  • weak RBS/LacZ alpha with EcoRI-HF+PstI
  • standard RBS/LacZ alpha/LuxI/term with EcoRI-HF+PstI
  • standard RBS/LacZ alpha with EcoRI-HF+PstI
  • -
  • attC (miniprep from 08/02)

Ge0208 3.jpg
This experiment indicates that the plasmid containing LuxI+term is not good (the length of the insert is 200bp instead of 700bp !)


Raphaël


Transformation of Turbo cells with yesterday's ligations using the Turbo cell transformation protocol:

  • stand.RBS/TetA(C)/LuxI/term/pLux/GFP
  • med.RBS/TetA(C)/LuxI/term/pLux/GFP
  • weak.RBS/TetA(C)/LuxI/term/pLux/GFP
  • stand.RBS/LacZα/LuxI/term/pLux/GFP
  • weakRBS/LacZα/LuxI/term/pLux/GFP
  • control 1 : pLux/GFP (vector only)
  • control 2 : stand.RBS/TetA(C)/LuxI/term (insert only)


Making Amp plates
30 plates


DNA quantification in the minipreps. Measured with the Nanodrop

Sample name 260/280 260/230 ng/µl
attC/term/attC/mRFP #1 2.10 2.26 148.2
attC/term/attC/mRFP #2 2.01 2.13 95.6
attC/term/attC/mRFP #3 2.06 2.17 105.1
attC/term/attC/mRFP #4 2.06 2.17 91.1
attC/term/attC/mRFP #5 2.06 2.17 140.5
attC/term/attC/mRFP #6 2.09 2.06 109.9
attC/term/attC/mRFP #7 2.09 1.94 71.5
attC/term/attC/mRFP #8 2.10 2.18 85.9
attC/term/attC/mRFP #9 2.07 2.11 104.3
attC/term/attC/mRFP #10 1.72 0.76 36.3
attC/term/attC/mRFP #11 1.61 0.93 33.4
attC/term/attC/mRFP #12 2.08 2.06 135.5
attC/term/attC/mRFP #13 2.02 1.76 139.4
attC/term/attC/mRFP #14 2.09 2.03 96.8
attC/term/attC/mRFP #15 2.05 1.85 142.7
attC/term/attC/mRFP #16 2.07 2.00 147.4
attC 2.11 2.03 130.5
term/attC/mRFP 2.15 2.34 130.4