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 (agarose 1.5% w/v, 50V)

  • 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. However, the transformation of pLux-RBS/GFP/term from 07/22 didn't work (see the gel made by Théotime further on this page)


Gel extraction
Elution from the column with 30µl of warm water (50°C). Samples of the same part are collected into 1 tube.


Nanodrop quantification of DNA amounts in the gel extraction samples

Sample name 260/280 260/230 ng/µl
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


Théotime
Restriction digest of the biobricks we might need again ifthe transformations don't work
20µl miniprep + 2.5µl Buffer 10x + 0.25 µl BSA 100x + 1µl enzyme1 + 1µl enzyme2 (total of 25µl)

  • RBS/GFP/term (E0240) (XbaI + PstI, B3)
  • RBS/LuxI (K081008) (EcoRI-HF + SpeI, B4)


40µl miniprep + 4.7µl Buffer 10x + 0.5 µl BSA 100x + 2µl enzyme1 + 2µl enzyme2 (total of 50µl) = double

  • LuxR (C0062) (XbaI + PstI, B3)
  • LacZ alpha (XbaI + PstI, B3)
  • TetA(C) (XbaI + PstI, B3)


Gel electrophoresis (1.5% agarose w/v, 50V)
20µl digestion product + 5µl lb 6x

  • ladder 1kb
  • RBS/GFP/term
  • -
  • TetA(C)
  • TetA(C)
  • -
  • LuxR
  • LuxR
  • -
  • -
  • -
  • pLux-GFP (ligation product purified by Aleksandra and Stéphane)


Gel electrophoresis (3.0% agarose w/v, 50V)
20µl digestion product + 5µl lb 6x

  • ladder 0.1kb
  • -
  • LacZ alpha
  • LacZ alpha
  • -
  • LuxI


All of the restictions worked (except for the verification of the ligation pLux-GFP).


Gel extraction>/u>
Elution from the column with 30µl of warm water (50°C). Samples of the same part are collected into 1 tube.


Nanodrop quantification of DNA amounts in the gel extraction samples

Sample name 260/280 260/230 ng/µl
LacZ alpha 1.94 0.02 9.0
LuxI 1.82 0.05 10.2
LuxR 1.87 0.13 36.6
TetA(C) 1.88 0.37 71.8
RBS/GFP/term 1.72 0.15 15.1


Ligation using around 150ng of the insert and 50µg of the vector, in 10µl of total volume if possible, or in a minimum volume if >10µl. Incubation at 16°C overnight.

  • 2µl TetA(C) + 0.9
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