IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/10

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Raphaël and Théotime
Minipreps, elution with 35µl warm water :

  • 11 attC/term/attC/mRFP
  • Pst/weakRBS/LuxR
  • Pst/medRBS/LuxR
  • Pmed/weakRBS/LuxR
  • Pmed/medRBS/LuxR
  • Pmed/stRBS/LuxR
  • Pmed
  • Pst
  • wRBS/TetA(C)
  • medRBS/TetA(C)


Nanodrop

Sample name 260/280260/230 ng/µl
attC/term/attC/mRFP2.081.88 119.5
Pmed/wRBS/LuxR 2.00 1.98 162.0
Ps/wRBS/LuxR 1.75 1.67 140.4
Pm/mRBS/luxR 1.97 1.94 265.9
Ps/mRFP/LuxR 2.06 1.79 117.6
Pm/sRBS/LuxR 1.97 1.88 188.4


Restriction digest of today's minipreps

  • Pst/weakRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes
  • Pst/medRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes
  • Pmed/weakRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes
  • Pmed/medRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes
  • Pmed/stRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes
  • wRBS/TetA(C) with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes
  • medRBS/TetA(C) with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes


Restriction digest of previuos minipreps:

  • Pst/stRBS/LuxR with SpeI and PstI : 20µl miniprep + 2.5µl B2 + 0.25 µl BSA + 2x1µl enzymes
  • stRBS/TetA(C) with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes
  • stLacZα with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes
  • K081014 col2 with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes
  • I13207 col2 with SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes
  • K081014 with EcoRI-HF and XbaI : SpeI and EcoRI-HF : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes
  • pLux/RBS with EcoRI-HF and XbaI : 20µl miniprep + 2.5µl B3 + 0.25 µl BSA + 2x1µl enzymes


Gel electrophoresis
Gel 1

  • attC/term/attC/mRFP in a big well
  • -
  • weakRBS/TetA(C)
  • -
  • medRBS/TetA(C)
  • -
  • stRBS/TetA(C)
  • Ladder 1kb

Image:gel1008_1.jpg Image:gel1008_1cut.jpg


Gel 2

  • stRBS/LacZα
  • Ladder 100bp
  • K081014
  • -
  • I13507
  • Ladder 1kb

Image:gel1008_2.jpg Image:gel1008_2cut.jpg


Aleksandra
Gel extraction
Elution in 35µl warm water

  • attC/term/attC/mRFP
  • weakRBS/TetA(C)
  • medRBS/TetA(C)
  • stRBS/TetA(C)
  • stRBS/LacZα
  • K081014
  • I13507


Raphaël
PCR purification kit

  • Pm/wRBS/LuxR
  • Ps/wRBS/LuxR
  • Pm/mRBS/luxR
  • Ps/mRBS/LuxR
  • Pm/sRBS/LuxR
  • Ps/sRBS/LuxR
  • LuxI
  • pLux/GFP


Nanodrop

Sample name 260/280260/230 ng/µl
mRBS/TetA(C) 1.70 0.07 6.6
wRBS/tetA(C) 1.41 0.07 5.1
sRBS/TetA(C) 2.01 0.07 7.3
sRBS/LacZα 1.59 0.05 3.0
K081014 2.31 0.11 7.4
I13507 2.04 0.087.5
attC/term/attC/mRFP 1.58 0.07 12.8
pLux/GFP 1.80 1.51 45.8
Pm/wRBS/LuxR 1.71 1.2131.5
Ps/wRBS/LuxR 1.97 0.68 8.7
Pm/mRBS/luxR 1.81 1.46 44.4
Ps/mRBS/LuxR 1.42 0.51 15.9
Pm/sRBS/LuxR 1.77 0.9425.8
Ps/sRBS/LuxR 1.37 0.39 5.2
Ps 1.580.64 26.6


Ligation

  • mRBS/TetA(C)+RBS/LuxI : 10µl + 1µl + 1.2µl buffer + 0.5µl ligase
  • wRBS/TetA(C)+RBS/LuxI : 10µl + 1µl + 1.2µl buffer + 0.5µl ligase
  • stRBS/TetA(C)+RBS/LuxI : 10µl + 1µl + 1.2µl buffer + 0.5µl ligase
  • stRBS/LacZα+RBS/LuxI : 10µl + 1µl + 1.2µl buffer + 0.5µl ligase
  • RBS/mRFP/t(K081014)+pLux/GFP : 5µl + 1µl(1/4dilution) + 0.7µl buffer + 0.5µl ligase
  • RBS/mRFP/tt(I13507)+pLux/GFP : 5µl + 1µl(1/4dilution) + 0.7µl buffer + 0.5µl ligase
  • Pm/wRBS/LuxR +attC/term/attC/mRFP : 1µl (1/3dilutuion) + 3µl + 1µl buffer + 0.5µl ligase + 4µl water
  • Ps/wRBS/LuxR +attC/term/attC/mRFP : 2µl + 3µl + 1µl buffer + 0.5µl ligase + 4µl water
  • Pm/mRBS/luxR +attC/term/attC/mRFP : 1µl (1/4dilutuion) + 3µl + 1µl buffer + 0.5µl ligase + 4µl water
  • Ps/mRBS/LuxR +attC/term/attC/mRFP : 1µl + 3µl + 1µl buffer + 0.5µl ligase + 4µl water
  • Pm/sRBS/LuxR +attC/term/attC/mRFP : 1µl (1/2dilutuion) + 3µl + 1µl buffer + 0.5µl ligase + 4µl water
  • Ps/sRBS/LuxR +attC/term/attC/mRFP : 2µl + 3µl + 1µl buffer + 0.5µl ligase + 4µl water


Controls:

  • RBS/LuxI : 10µl water + 1µl + 1.2µl buffer + 0.5µl ligase
  • pLux/GFP : 5µl water + 1µl(1/4dilution) + 0.7µl buffer + 0.5µl ligase
  • Pm/wRBS/LuxR : 1µl (1/3dilutuion) + 1µl buffer + 0.5µl ligase + 7µl water
  • Ps/wRBS/LuxR : 2µl + 1µl buffer + 0.5µl ligase + 7µl water
  • Pm/mRBS/luxR : 1µl (1/4dilutuion)+ 1µl buffer + 0.5µl ligase + 7µl water
  • Ps/mRBS/LuxR : 1µl + 1µl buffer + 0.5µl ligase + 7µl water
  • Pm/sRBS/LuxR : 1µl (1/2dilutuion) + 1µl buffer + 0.5µl ligase + 7µl water
  • Ps/sRBS/LuxR : 2µl + 1µl buffer + 0.5µl ligase + 7µl water


Aleksandra
Overnight cultures in 10ml cultures

  • Pst/weakRBS/LuxR
  • Pst/sRBS/LuxR
  • Pmed
  • wRBS/TetA(C)
  • medRBS/TetA(C)
  • sRBS/TetA(C)
  • wRBS/LacZα
  • sRBS/LacZα


Plating
As some of our overnight cultures had no bvacteria, we decided to plate the glycerols to verify if they work and do some new glycerols.

  • Ps/sRBS/LuxR
  • wRBS/LacZα
  • stRBS/LacZα
  • stRBS/TetA(C)



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