IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/09/02

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Aleksandra
Gel extraction

  • Ps/sRBS/LuxR/attC/term/attC/mRFP
  • Pm/sRBS/LuxR/attC/term/attC/mRFP
  • I13507/pLux/GFP
  • wRBS/tetA(C)
  • mRBS/tetA(C)
  • sRBS/tetA(C)
  • sRBS/LacZα/LuxI III (from )
  • pLux/LuxI II
  • Ps/mRBS/LuxR/attC/term/attC/wRBS/TetA(C) II
  • Ps/mRBS/LuxR/attC/term/attC/wRBS/TetA(C) III


Raphaël
Minipreps

  • attP-E (pSWK)
  • Pm/sRBS/LuxR/attC/term/attC/mRFP


Restriction digest
With EcoRI-HF and PstI (15µl DNA + 2µl B2 + 2x1µl enzymes + 0.2µl BSA):

  • attP-E (pSWK)


With EcoRI-HF and PstI and AlWNI (10µl DNA + 5µl water + 2µl B2 + 3x1 µl enzyme + 0.2µl BSA)

  • Ps/wRBS/LuxR/attC/term/attC/mRFP
  • Ps/mRBS/LuxR/attC/term/attC/mRFP
  • Ps/sRBS/LuxR/attC/term/attC/mRFP
  • Pm/sRBS/LuxR/attC/term/attC/mRFP


Gel electrophoresis

  • attP-E (pSWK)
  • -
  • Ps/wRBS/LuxR/attC/term/attC/mRFP
  • -
  • Ps/mRBS/LuxR/attC/term/attC/mRFP
  • -
  • Ps/sRBS/LuxR/attC/term/attC/mRFP
  • -
  • Pm/sRBS/LuxR/attC/term/attC/mRFP
  • -
  • Ladder 1 kb
  • -
  • -
  • -
  • -
  • -
  • -

File:Gel0209 1.jpg


Gel extraction

  • attP-E (pSWK)
  • Ps/mRBS/LuxR/attC/term/attC/mRFP
  • Ps/sRBS/LuxR/attC/term/attC/mRFP
  • Pm/sRBS/LuxR/attC/term/attC/mRFP


Ligations (V + I)

  • 1) attP + Pms
  • 2) attP + Psm
  • 3) attP + Pss
  • 4) pLux + LuxI
  • 5) wlacZα + LuxI
  • 6) sLacZα + LuxI
  • 7) Psm + slacZα/LuxI III
  • 8) Psm + wTet/LuxI
  • 9) Pss + wTet/LuxI
  • 10) Pms + wTet/LuxI
  • 11) I13507/pLux/GFP + Psm/wTet II
  • 12) I13507/pLux/GFP + Psm/wTet III
  • 13) mTet/LuxI + Psm
  • 14) sTet/LuxI + Psm
  • 15) sLacZα/LuxI III + Pss
  • 16) mTet/LuxI + Pss
  • 17) sTet/LuxI + Pss
  • 18) sLacZα/LuxI III + Pms
  • 19) mTet/LuxI + Pms
  • 20) sTet/LuxI + Pms
  • A) ctrl attP
  • B) ctrl pLux
  • C) ctrl wLacZα
  • D) ctrl sLacZα
  • E) ctrl Psm
  • F) ctrl Pss
  • G) ctrl Pms
  • H) ctrl I13507


Aleklsandra
Transformation of 1-20, A-H, plating with 150 to 200µl


Overnight cultures

  • Ps/wRBS/LuxR/attC/term/attC/mRFP and pBAD/Int (double transformants)
  • Ps/mRBS/LuxR/attC/term/attC/mRFP and pBAD/Int (double transformants)
  • Ps/sRBS/LuxR/attC/term/attC/mRFP and pBAD/Int (double transformants)
  • wRBS/tetA(C)
  • mRBS/tetA(C)
  • sRBS/tetA(C)
  • wRBS/LacZα
  • sRBS/LacZα
  • Ps