IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/26: Difference between revisions

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<font color=red>Antoine</font>
Electrophoresis of digest products


* Insert your content here.
<br><u>Restriction digest</u>
 
{|
|Sample name||DNA (3ug)||enzyme||Buffer 2||H2O||BSA
|-
|C0040  || 9.4 ||1 X/P ||2.5||10.85||0.25
|-
|C0012  || 19.4 ||1 X/P ||2.5||0.85||0.25
|-
|B0015 || 20.25||1 X/P ||2.5||0||0.25
|-
|J23110 || 20.25||1 S/P ||2.5||0||0.25
|-
|P1003 || 6||1 S/P ||2.5||14.25||0.25
|}
|-
|-
|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> Population counter </span>
|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> Population counter </span>
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*strong RBS-LuxR
*strong RBS-LuxR


[[Image:gel2307.jpg]]
[[Image:gel2307.jpg]][[Image:gel23072.jpg]]


<br>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.
<br>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.
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<font color=red>Théotime</font>
<font color=red>Théotime and Raphaël</font>
<br><u>Restriction digest of the biobricks we might need again ifthe transformations don't work</u>
<br><u>Restriction digest of the biobricks we might need again if the transformations don't work</u>
<br>20µl miniprep + 2.5µl Buffer 10x + 0.25 µl BSA 100x + 1µl enzyme1 + 1µl enzyme2 (total of 25µl)
<br>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/GFP/term (E0240) (XbaI + PstI, B3)
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*pLux-GFP (ligation product purified by Aleksandra and Stéphane)
*pLux-GFP (ligation product purified by Aleksandra and Stéphane)


[[Image:gel2607_2.jpg]]
[[Image:gel2607_2.jpg]][[Image:gel2607_22.jpg]]


<br><u>Gel electrophoresis (3.0% agarose w/v, 50V)</u>
<br><u>Gel electrophoresis (3.0% agarose w/v, 50V)</u>
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*LuxI
*LuxI


[[Image:gel2607_3.jpg]]
[[Image:gel2607_3.jpg]] [[Image:gel2607_32.jpg]]


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


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


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<br><u>Ligation</u> 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.
<br><u>Ligation</u> 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) + 0.9
*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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Revision as of 07:57, 2 August 2010

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Antoine Electrophoresis of digest products


Restriction digest

Sample name DNA (3ug) enzyme Buffer 2 H2O BSA
C0040 9.4 1 X/P 2.5 10.85 0.25
C0012 19.4 1 X/P 2.5 0.85 0.25
B0015 20.25 1 X/P 2.5 0 0.25
J23110 20.25 1 S/P 2.5 0 0.25
P1003 6 1 S/P 2.5 14.25 0.25
Population counter <html><img src="/images/9/94/Report.png" border="0" /></html> 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


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 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)


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
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. 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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