Team:Paris Liliane Bettencourt/Notebook/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)

<a href="/wiki/Image:Gel0208_1.jpg" class="image" title="Image:gel0208_1.jpg"><img alt="Image:gel0208_1.jpg" src="/images/9/93/Gel0208_1.jpg" width="768" height="574" border="0" /></a>
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

<a href="/wiki/Image:Gel0208_2.jpg" class="image" title="Image:gel0208_2.jpg"><img alt="Image:gel0208_2.jpg" src="/images/2/21/Gel0208_2.jpg" width="768" height="574" border="0" /></a>


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)

<a href="/wiki/Image:Ge0208_3.jpg" class="image" title="Image:ge0208_3.jpg"><img alt="Image:ge0208_3.jpg" src="/images/a/a1/Ge0208_3.jpg" width="768" height="574" border="0" /></a>
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 name260/280260/230ng/µl
attC/term/attC/mRFP #12.10 2.26 148.2
attC/term/attC/mRFP #22.012.13 95.6
attC/term/attC/mRFP #32.062.17105.1
attC/term/attC/mRFP #42.062.17 91.1
attC/term/attC/mRFP #52.062.17140.5
attC/term/attC/mRFP #62.092.06109.9
attC/term/attC/mRFP #72.091.94 71.5
attC/term/attC/mRFP #82.102.18 85.9
attC/term/attC/mRFP #92.072.11 104.3
attC/term/attC/mRFP #10 1.720.76 36.3
attC/term/attC/mRFP #11 1.610.93 33.4
attC/term/attC/mRFP #122.082.06 135.5
attC/term/attC/mRFP #132.021.76 139.4
attC/term/attC/mRFP #142.092.0396.8
attC/term/attC/mRFP #152.051.85142.7
attC/term/attC/mRFP #162.072.00147.4
attC2.112.03130.5
term/attC/mRFP2.152.34130.4
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