Team:Paris Liliane Bettencourt/Notebook/2010/08/15/

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Raphaël


Restriction digest , 2h at 37°C


We want to amplify this DNA, so the idea is to cut the plasmid once to obtain a linear fragment that can be easily amplified with our primers.

  • mRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI
  • wRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI
  • stRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI


PCR
Primers : VF2 + VR

  • mRBS/TetA(C)+RBS/LuxI
  • wRBS/TetA(C)+RBS/LuxI
  • sRBS/TetA(C)+RBS/LuxI
  • mRBS/TetA(C)+RBS/LuxI cut by AlwNI
  • wRBS/TetA(C)+RBS/LuxI cut by AlwNI
  • sRBS/TetA(C)+RBS/LuxI cut by AlwNI


-> PCR tubes stored overnight at -20°C.


Ligation for 1h30 at 25°C

  • wLac+LuxI : 1µl 1/2 dilution + 4µl + 1µL buffer + 4µL H20 + 0.5µl ligase
  • sLac+LuxI : 1µl 1/4 dilution + 4µl + 1µL buffer + 4µL H20 + 0.5µl ligase


Control with only LuxI has already be done yesterday.


Transformation in Turbocells


With today's ligations:

  • wLac+LuxI
  • sLac+LuxI

Control :

  • positive control (pUC19)


Overnight cultures
From glycerols :

  • wRBS/LacZα
  • sRBS/LacZα
  • RBS/LuxI
  • pLux
  • attC-t-attC-mRFP
  • PmM-LuxR
  • PmW-LuxR
  • wTet-LuxI
  • mTet-LuxI
  • sTet-LuxI


From Aleksandra's transformations :

  • 1x PmW-LuxR-attC
  • 3x pLux-LuxI
  • 4x PsS-attC + pBad-Int
  • 4x PsM-attC + pBad-Int
  • 4x PsW-attC + pBad-Int
  • 4x PmS-attC + pBad-Int