IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/21

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Memo cell Main project page
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Raphaël


Gel extraction

  • Left arm HK022 Mut-Mut
  • Left arm HK022 WT-Mut
  • Left arm λ Mut-Mut
  • Left arm λ WT-Mut
  • Left arm WT-WT
  • Right arm HK022 Mut-Mut
  • Right arm HK022 WT-Mut
  • Right arm λ Mut-Mut
  • Right arm λ WT-Mut


PCR purification

  • Kan resistance cassette
  • LacZα expression cassette


Ligations

  • 1)Left arm HK022 Mut-Mut + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 2)Left arm HK022 WT-Mut + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 3)Left arm λ Mut-Mut + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 4)Left arm λ WT-Mut + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 5)Left arm WT-WT + Kan resistance cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 6)Right arm HK022 Mut-Mut + LacZα expression cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 7)Right arm HK022 WT-Mut + LacZα expression cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 8)Right arm λ Mut-Mut + LacZα expression cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 9)Right arm λ WT-Mut + LacZα expression cassette (6µl + 1µl + 0.5µl ligase + 1µl buffer + 3µl water)


Controls:

  • 13)Kan resistance cassette (1µl + 0.5µl ligase + 1µl buffer + 9µl water)
  • 14)LacZα expression cassette (1µl + 0.5µl ligase + 1µl buffer + 9µl water)


Transformations of DH5α with 2µl of miniprep, plating with 150µl

  • Left arm HK022 Mut-Mut
  • Left arm HK022 WT-Mut
  • Left arm λ Mut-Mut
  • Left arm λ WT-Mut
  • Left arm WT-WT
  • Right arm HK022 Mut-Mut
  • Right arm HK022 WT-Mut
  • Right arm λ Mut-Mut
  • Right arm λ WT-Mut
  • 17)pUC19 (positive control)


Population counter Main project page
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Raphaël


Restriction digest with SpeI and PstI : 10µl miniprep + 10µL water + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA

  • Ps/wRBS/LuxR + attC/term/attC/mRFP
  • Ps/mRBS/LuxR + attC/term/attC/mRFP


Restriction digest with XbaI and PstI : 10µl miniprep + 10µL water + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA

  • wRBS/TetA(C)
  • mRBS/TetA(C)
  • RBS/LuxI


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