IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/08/03

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Théotime and Raphaël
Transformation of TURBO cells, incubation at 37°C around 10h

  • RBS/LuxI/term (ligation of 26/07), with 50µl
  • RBS/LuxI/term (ligation of 26/07), with 100µl
  • positive control


Aleksandra
Minipreps of 10ml, elution in 35µl warm water

  • attC (from glycerol stock)
  • term/attC/mRFP (from glycerol stock)
  • attC/term/attC/mRFP #10 (from yesterday's glycerol stock)
  • attC/term/attC/mRFP #11 (from yesterday's glycerol stock)


DNA quantification in the minipreps using the nanodrop

Sample name 260/280 260/230 ng/µl
attC 2.13 1.69 135.2
term 2.13 2.07 169.9
attC/term/attC/mRFP #10 2.02 1.91 118.0


Léa and Aleksandra
Digestion

  • attC (today's miniprep) with SpeI+PstI (B2) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA
  • term/attC/mRFP (today's miniprep) with XbaI+PstI (B3) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA
  • 16 minipreps attC/term/attC/mRFP (yesterday's and today's minipreps) with EcoRI-HF + SpeI (B4) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA
  • RBS/LuxI/term (miniprep from 26/07) with EcoRI-HF + SpeI (B4) : 10µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA + 6µl water
  • RBS/LuxI (miniprep from 24/07) with EcoRI-HF + SpeI (B4) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA
  • term (miniprep from 24/07) with EcoRI-HF + SpeI (B4) : 20µl miniprep + 2x1µl enzyme + 2µl buffer + 0.2µl BSA


Gel electrophoresis, 15%agarose, no EtB
Gel 1, 75V

  • attC/term/attC #1
  • -
  • attC/term/attC #2
  • ladder 100bp
  • attC/term/attC #3
  • -
  • attC/term/attC #4
  • -
  • attC/term/attC #5
  • -
  • attC/term/attC #6
  • -
  • attC/term/attC #7
  • -
  • attC/term/attC #8
  • -
  • attC/term/attC #9

Gel1 0308.jpg


Gel 2, 75V

  • attC/term/attC #10
  • -
  • attC/term/attC #11
  • -
  • -
  • attC/term/attC #12
  • -
  • attC/term/attC #13
  • -
  • attC/term/attC #14
  • ladder 1kb
  • attC/term/attC #15

Gel2 0308.jpg Gel2 0308cut.jpg
#10 and #11 have worked!


Gel 3, 50V

  • attC/term/attC #16
  • attC
  • -
  • term/attC/mRFP
  • -
  • RBS/LuxI/term
  • -
  • RBS/luxI
  • -
  • ladder 100bp
  • -
  • ladder 1kb

Gel3 0308.jpg Gel3 0308cut.jpg


Gel extraction, elution in 30µl warm water

  • RBS/LuxI
  • attC/term/attC #10
  • attC/term/attC #11


PCR kit purification

  • term


DNA quantification after gel extraction and PCR purification kit using the nanodrop

Sample name 260/280 260/230 ng/µl
RBS/LuxI
attC/term/attC #10
attC/term/attC #11
term


Léa
Ligation

  • 25µl LuxI miniprep + 2µl term miniprep + 1µl ligase + 3µl buffer
  • 2µl term miniprep + 1µl ligase + 1µl buffer + 6µl water (control)


Léa, Raphaël and Aleksandra
Transformation of TOP10 cells, incubation at 37°C overnight

  • 25µl LuxI miniprep
  • 2µl term miniprep


Raphaël and Aleksandra
Overnight cultures, 10ml with Amp

  • Pm
  • Ps
  • Pm/weakRBS/LuxR
  • Ps/weakRBS/LuxR
  • Pm/medRBS/LuxR
  • Ps/medRBS/LuxR
  • Pm/standRBS/LuxR
  • Ps/standRBS/LuxR
  • RBS/LuxI
  • term


Overnight cultures, 5x20ml with Kan

  • attC/term/attC/mRFP #10


Aleksandra
Overnight cultures started from this morning's TURBO cell plates, 2 colonies per plate

  • RBS/LuxI/term