IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/16

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Aleksandra
Restriction digest with XbaI and SacI : 3µl miniprep + 2x1µl enzymes + 2µl B4 + 0.2µl BSA + 12.8µl water

  • SacI-E-X-J23110/RBS/attP HK wt1-S-P-SacI
  • SacI-E-X-J23110/RBS/attP HK wt2-S-P-SacI
  • SacI-E-X-J23110/RBS/attP HK mut1-S-P-SacI
  • SacI-E-X-J23110/RBS/attP HK mut2-S-P-SacI
  • SacI-E-X-J23110/RBS/attP λ mut1-S-P-SacI
  • SacI-E-X-J23110/RBS/attP λ mut2-S-P-SacI


Restriction digest with SpeI and SacI : 18µl miniprep + 2x3µl enzymes + 6µl B1 + 0.6µl BSA + 30µl water

  • S-P-SacI-attP-SacI
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Léa
Glycerols and minipreps

  • wRBS/LacZα
  • sRBS/LacZα
  • LuxI
  • pLux
  • attC/t/attC/mRFP
  • Pm/mRBS/LuxR
  • Pm/wRBS/LuxR
  • wRBS/TetA(C)/LuxI
  • mRBS/TetA(C)/LuxI
  • sRBS/TetA(C)/LuxI
  • Pm/wRBS/LuxR/attC/t/attC/mRFP
  • pLux/LuxI (started with 3 colonies)


Nanodrop of the minipreps

Sample name 260/280 230/280 ng/µl
wRBS/LacZα 1.92 2.34 128.7
sRBS/LacZα 1.91 1.70 175.1
pLux 1.90 2.07 222.9
attC/t/attC/mRFP 1.88 1.91 219.0
Pm/mRBS/LuxR 1.93 2.35 686.8
Pm/wRBS/LuxR 1.87 2.15 437.8
wRBS/TetA(C)/LuxI 1.96 2.27 235.4
mRBS/TetA(C)/LuxI 1.95 2.19 282.6
sRBS/TetA(C)/LuxI 1.91 2.26 340.2


Léa
Restriction digest with SpeI and PstI : 20µl miniprep + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA

  • wRBS/LacZα
  • sRBS/LacZα
  • pLux
  • Pm/mRBS/LuxR
  • Pm/wRBS/LuxR
  • Pm/wRBS/LuxR/attC/t/attC/mRFP (5µl miniprep + 2x1µl enzyme + 2.5µl B2 + 0.25µl BSA + 15µl water)


Restriction digest with XbaI and PstI :

  • LuxI (20µl miniprep + 2x1µl enzyme + 2.5µl B3 + 0.25µl BSA)
  • attC/t/attC/mRFP (20µl miniprep + 2x1µl enzyme + 2.5µl B3 + 0.25µl BSA)
  • pLux/LuxI, 3 different minipreps (5µl miniprep + 2x1µl enzyme + 2.5µl B3 + 0.25µl BSA + 15µl water)


Aleksandra
Gel electrophoresis

  • wRBS/TetA(C)/LuxI (PCR of the uncut plasmid)
  • mRBS/TetA(C)/LuxI (PCR of the uncut plasmid)
  • sRBS/TetA(C)/LuxI (PCR of the uncut plasmid)
  • Ladder 1kb
  • wRBS/TetA(C)/LuxI (PCR of the plasmid cut by AlWNI), 2 wells
  • -
  • mRBS/TetA(C)/LuxI (PCR of the plasmid cut by AlWNI), 2 wells
  • -
  • sRBS/TetA(C)/LuxI (PCR of the plasmid cut by AlWNI), 2 wells

File:Gel1608 1.jpg File:Gel1608 1cut.jpg


Gel extraction

  • wRBS/TetA(C)/LuxI (PCR of the plasmid cut by AlWNI)
  • mRBS/TetA(C)/LuxI (PCR of the plasmid cut by AlWNI)
  • sRBS/TetA(C)/LuxI (PCR of the plasmid cut by AlWNI)


Nanodrop of the gel extractions

Sample name 260/280 230/280 ng/µl
wRBS/TetA(C)/LuxI 1.91 0.32 47.9
mRBS/TetA(C)/LuxI 1.87 0.25 40.7
sRBS/TetA(C)/LuxI 1.87 0.35 63.0


Restriction digest of the with XbaI and PstI : 20µl miniprep + 2x1µl enzyme + 2.5µl B3 + 0.25µl BSA

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


PCR kit purification, elution in 35µl warm water

Sample name 260/280 230/280 ng/µl
wRBS/TetA(C)/LuxI 1.68 1.40 19.8
mRBS/TetA(C)/LuxI 1.59 0.96 21.6
sRBS/TetA(C)/LuxI 1.73 1.54 28.2
pLux 1.75 1.37 62.7


Ligations

  • 1)Pm/sRBS/LuxR + wRBS/TetA(C)/LuxI (0.5µl + 4.5µl + 0.5µl ligase + 1µl buffer + 3.5µl water)
  • 2)Ps/wRBS/LuxR + wRBS/TetA(C)/LuxI (1µl + 4.5µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 3)Ps/mRBS/LuxR + wRBS/TetA(C)/LuxI (1µl + 4.5µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 4)Ps/sRBS/LuxR + wRBS/TetA(C)/LuxI (0.5µl + 4.5µl + 0.5µl ligase + 1µl buffer + 3.5µl water)
  • 5)Pm/sRBS/LuxR + mRBS/TetA(C)/LuxI (0.5µl + 4.5µl + 0.5µl ligase + 1µl buffer + 3.5µl water)
  • 6)Ps/wRBS/LuxR + mRBS/TetA(C)/LuxI (1µl + 4.5µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 7)Ps/mRBS/LuxR + mRBS/TetA(C)/LuxI (1µl + 4.5µl + 0.5µl ligase + 1µl buffer + 3µl water)
  • 8)Ps/sRBS/LuxR + mRBS/TetA(C)/LuxI (0.5µl + 4.5µl + 0.5µl ligase + 1µl buffer + 3.5µl water)
  • 9)Pm/sRBS/LuxR + sRBS/TetA(C)/LuxI (0.5µl + 4.5µl + 0.5µl ligase + 1µl buffer + 5µl water)
  • 10)Ps/wRBS/LuxR + sRBS/TetA(C)/LuxI (1µl + 4.5µl + 0.5µl ligase + 1µl buffer + 4.5µl water)
  • 11)Ps/mRBS/LuxR + sRBS/TetA(C)/LuxI (1µl + 4.5µl + 0.5µl ligase + 1µl buffer + 4.5µl water)
  • 12)Ps/sRBS/LuxR + sRBS/TetA(C)/LuxI (0.5µl + 4.5µl + 0.5µl ligase + 1µl buffer + 5µl water)


Controls:

  • 13)Pm/sRBS/LuxR (0.5µl + 0.5µl ligase + 1µl buffer + 8µl water)
  • 14)Ps/wRBS/LuxR (1µl + 0.5µl ligase + 1µl buffer + 7.5µl water)
  • 15)Ps/mRBS/LuxR (1µl + 0.5µl ligase + 1µl buffer + 7.5µl water)
  • 16)Ps/sRBS/LuxR (0.5µl + 0.5µl ligase + 1µl buffer + 8µl water)


Raphaël
Glycerols

  • Ps/sRBS/LuxR/attC/t/attC/mRFP and pBAD/IntI, 4 cultures
  • Ps/wRBS/LuxR/attC/t/attC/mRFP and pBAD/IntI, 4 cultures
  • Ps/mRBS/LuxR/attC/t/attC/mRFP and pBAD/IntI, 4 cultures
  • Pm/sRBS/LuxR/attC/t/attC/mRFP and pBAD/IntI, 4 cultures
  • pLux/RBS/LuxI, 3 cultures
  • Pm/wRBS/attC/t/attC/mRFP, 1 cultures


Minipreps

  • LuxI
  • Pm/wRBS/LuxR/attC/t/attC/mRFP
  • pLux/LuxI, 3 cultures


Nanodrop of the minipreps

Sample name 260/280 230/280 ng/µl
LuxI 1.97 2.25 244.2
Pm/wRBS/LuxR/attC/t/attC/mRFP 1.96 2.27 256.8
pLux/LuxI cult1 1.96 2.12 195.1
pLux/LuxI cult2 2.60 2.27 186.2
pLux/LuxI cult3 2.00 2.16 174.0


Overnight cultures

  • wRBS/LacZα/LuxI, 4 colonies
  • sRBS/LacZα/LuxI, 4 colonies
  • LuxI