IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/29

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Raphaël
Making Amp plates
40 plates

Aleksandra and Théotime
Miniprep

  • stand.RBS /TetA(C) 1 and 2
  • pLux/RBS/GFP/term A and B (transformations from 07/27)
  • Pmed/weakRBS/LuxR 1 and 2
  • Pstr/weakRBS/LuxR 1 and 2
  • Pmed /medRBS/LuxR 1 and 2
  • Pstr/medRBS/LuxR 1 and 2
  • Pmed/strRBS/LuxR 1 and 2
  • Pstr/strRBS/LuxR 1 and 2
  • stand.RBS/LacZ 1 and 2
  • weak RBS/LacZ 1 and 2
  • LuxI/term 1 and 2
  • attC
  • term/attC/mRFP
  • pLux/RBS/GFP/term 1 to 5 (transformations from 07/21)


Théotime and Raphaël
Restriction digest, incubation at 37°C for 2h.
Restriction digests to do gel purification :

  • 20µl stand.RBS /TetA(C) 1 + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x
  • 20µl med.RBS /TetA(C) + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x
  • 20µl weak.RBS /TetA(C) + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x
  • 20µl stand.RBS /LacZ 1 + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x
  • 20µl weak.RBS /LacZ 1 + 1µl EcoRI + 1µl SpeI + 2.5µl buffer4 10x + 0.25µl BSA 100x
  • 20µl LuxI/term 1 + 1µl EcoRI + 1µl XbaI + 2.5µl buffer4 10x + 0.25µl BSA 100x
  • 20µl attC + 1µl SpeI + 1µl PstI + 2.5µl buffer2 10x + 0.25µl BSA 100x
  • 20µl term/attC/mRFP + 1µl XbaI + 1µl PstI + 2.5µl buffer3 10x + 0.25µl BSA 100x


Restriction digests to verify the transformations :

  • 5µl pLux/GFP 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl pLux/GFP 2 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl pLux/GFP 3 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl pLux/GFP 4 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl pLux/GFP A + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl pLux/GFP B + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl Pstr/weakRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl Pmed/weakRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl Pstr/medRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl Pmed /medRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl Pstr/strRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water
  • 5µl Pmed/strRBS/LuxR 1 + 1µl XbaI + 1µl PstI + 2µl buffer3 10x + 0.2µl BSA 100x + 11µl water


Aleksandra and Raphaël
Gel electrophoresis (1.5% agarose, 100V) Since the yield of the gel purification was bad (between 5 and 30%), the experiment was done at the Necker's lab in order to control the role of materials and buffers.

  • pLux/GFP 1 : 20µl + 5µl lb
  • pLux/GFP 2 : 20µl + 5µl lb
  • pLux/GFP 3 : 20µl + 5µl lb
  • pLux/GFP 4 : 20µl + 5µl lb
  • pLux/GFP 5 : 20µl + 5µl lb
  • pLux/GFP A : 20µl + 5µl lb
  • pLux/GFP B : 20µl + 5µl lb
  • Pstr/weakRBS/LuxR 1 : 20µl + 5µl lb
  • Pmed/weakRBS/LuxR 1 : 20µl + 5µl lb
  • Pstr/medRBS/LuxR 1 : 20µl + 5µl lb
  • Pmed /medRBS/LuxR 1 : 20µl + 5µl lb
  • Ladder 1Kb (Fermentas) : 10 µL

Image:gel2907_1.jpg


  • Pstr/strRBS/LuxR 1 : 20µl + 5µl lb
  • Pmed/strRBS/LuxR 1 : 20µl + 5µl lb
  • Ladder 1Kb (Fermentas) : 10 µL
  • term/attC/mRFP : 25µl + 5µl lb
  • -
  • weak.RBS /TetA(C) 1 : 25µl + 5µl lb
  • -
  • med.RBS /TetA(C) 1 : 25µl + 5µl lb
  • -
  • stand.RBS /TetA(C) 1 : 25µl + 5µl lb
  • -
  • -

Image:gel2907_2.jpg Image:gel2907_3.jpg


  • weak.RBS /LacZ 1 : 25µl + 5µl lb
  • -
  • stand.RBS /LacZ 1  : 25µl + 5µl lb
  • -
  • Ladder 100bp : 10 µL
  • -

Image:gel2907_4.jpg Image:gel2907_5.jpg


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