IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/26

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Stéphane and Aleksandra
Verification of the transformations from 07/23 and 07/22 and if successful preparing them for future ligation
Restriction digest of the minipreps from 07/25 and 07/23, incubation at 37°C for 2h 20µl miniprep + 2.5µl Buffer3 10x + 0.25 µl BSA 100x + 1µl XbaI + 1µl PstI (total of 25µl)

  • weak RBS (B0031) - TetA(C) (J31007), transformation on 07/23
  • medium RBS (B0032) - TetA(C) (J31007), transformation on 07/23
  • pLux (R0062) - RBS/GFP/term (E0240), transformation on 07/22


40µl miniprep + 4.7µl Buffer3 10x + 0.5 µl BSA 100x + 2µl XbaI + 2µl PstI (total of 50µl) = double

  • weak RBS (B0031) - LuxR (C0062), transformation on 07/23
  • medium RBS (B0032) - LuxR (C0062), transformation on 07/23
  • strong RBS (B0034) - LuxR (C0062), transformation on 07/23


Gel electrophoresis

  • weak RBS-TetA(C)
  • ladder
  • medium RBS-TetA(C)
  • -
  • weak RBS-LuxR
  • weak RBS-LuxR
  • -
  • medium RBS-LuxR
  • medium RBS-LuxR
  • -
  • strong RBS-LuxR
  • strong RBS-LuxR


The transformations from 07/23 have worked, we have the inserts of the expected size : 1.2kb for RBS+TetA(C) and 0.8kb for RBS+LuxR. We can purify these inserts to use them in further ligations.


Gel extraction
Elution from the column with 30µl of warm water (50°C).


Nanodrop quantification of DNA amounts in the gel extraction samples

Sample name 260/280 260/230 ng/µl
LuxR 1.87 0.13 36.6
TetA(C) 1.88 0.37 71.8
RBS/GFP/term 1.72 0.15 15.1
weak RBS-TetA(C) 1.62 0.11 11.8
medium RBS-TetA(C) 1.90 0.09 22.8
weak RBS-LuxR 1.76 0.38 32.3
medium RBS-LuxR 1.89 0.08 36.8
standard RBS-LuxR 1.80 0.16 40.7
LacZ alpha 1.94 0.02 9.0
LuxI 1.82 0.05 10.2


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