IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/22

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Aleksandra
Gel electrophoresis

  • Ps/wRBS/LuxR/attC/t/attC/mRFP
  • -
  • Ps/mRBS/LuxR/attC/t/attC/mRFP

-

  • sRBS/LacZ (wrong enzymes, no interest)
  • -
  • wRBS/TetA(C)
  • -
  • mRBS/TetA(C)
  • -
  • LuxI
  • Ladder 1kb

File:Gel2208 1.jpg


Gel extraction, elution in 35µl warm water

  • LuxI


PCR
1µl miniprep + 2x1µl primers VF2 and VR (0.025ng/µl) + 10µl water + 13µl 2x Quick load mix; Tm=60°C, Telong=72°C (should have been 68°C)

  • wRBS/TetA(C)
  • mRBS/TetA(C)
  • sRBS/TetA(C)


PCR purification kit, elution in 35µl warm water

  • wRBS/TetA(C)
  • mRBS/TetA(C)
  • sRBS/TetA(C)



Digestion with XbaI and SpeI : 15µl PCR product + 2x1µl enzymes + 2µl B3 + 0.2µl BSA, then inactivation 10mins at 80°C

  • wRBS/TetA(C)
  • mRBS/TetA(C)
  • sRBS/TetA(C)


Overnight cultures

  • Ps/sRBS/LuxR/attC/t/attC/mRFP
  • Ps/mRBS/LuxR/attC/t/attC/mRFP
  • Ps/wRBS/LuxR/attC/t/attC/mRFP