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

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|Sample name||DNA (3ug)||enzyme||Buffer 2||H2O||BSA
|Sample name||DNA (3ug)||enzyme||Buffer 2||H2O||BSA
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|C0040  || 9.4 ||1 X/P ||2.5||
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|C0040  || 9.4 ||1 X/P ||2.5||10.85||0.25
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|medium RBS-TetA(C) ||1.90  ||0.09 ||22.8
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|C0012 || 19.4 ||1 X/P ||2.5||0.85||0.25
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| || || ||
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|B0015 || 20.25||1 X/P ||2.5||0||0.25
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|weak RBS-LuxR  ||1.76  ||0.38 ||32.3
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|J23110 || 20.25||1 S/P ||2.5||0||0.25
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|medium RBS-LuxR  ||1.89  ||0.08 ||36.8
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|P1003 || 6||1 S/P ||2.5||14.25||0.25
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| standard RBS-LuxR || 1.80 ||0.16 ||40.7
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Revision as of 10:55, 2 August 2010

Memo-Cell Main project page
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Electrophoresis of digest products


Restriction digest

Sample nameDNA (3ug)enzymeBuffer 2H2OBSA
C0040 9.4 1 X/P 2.510.850.25
C0012 19.4 1 X/P 2.50.850.25
B0015 20.251 X/P 2.500.25
J23110 20.251 S/P 2.500.25
P1003 61 S/P 2.514.250.25
Population counter Main project page
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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

Image:gel2307.jpgImage:gel23072.jpg


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 name260/280260/230ng/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 and Raphaël
Restriction digest of the biobricks we might need again if the 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)

Image:gel2607_2.jpgImage:gel2607_22.jpg


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

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

Image:gel2607_3.jpg Image:gel2607_32.jpg


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


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 name260/280260/230ng/µl
LacZ alpha 1.94 0.029.0
LuxI1.82 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. All of the vector DNA (RBSs, Pm, Ps) are diluted 1/5. Incubation at 16°C overnight.

  • 2µl TetA(C) + 0.9µl stand.RBS + 1µl buffer + 0.5µl ligase + 5.5µl water
  • 10µl RBS/GFP/term + 3.6µl pLux + 1µl buffer + 0.5µl ligase + 4µl water
  • 5µl weakRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase
  • 5µl weakRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 0.8µl water
  • 4µl medRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase + 1µl water
  • 4µl medkRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 1.8µl water
  • 4µl strRBS/LuxR + 3.6µl Pmed + 1µl buffer + 0.5µl ligase + 1µl water
  • 4µl strRBS/LuxR + 2.7µl Pstr + 1µl buffer + 0.5µl ligase + 1.8µl water
  • 13µl LacZ + 0.7µl weak RBS + 2µl buffer + 0.5µl ligase + 3.8µl water
  • 13µl LacZ + 0.9µl stand RBS + 2µl buffer + 0.5µl ligase + 3.6µl water
  • 15µl LuxI + 2.9µl weak RBS + 2µl buffer + 0.5µl ligase



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