Team:Evry/Notebook/w8

From 2013.igem.org

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<h1>Week 8: 5th August - 11th August</h1>
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<h2>Plasmide 3:</h2>
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<h1>Week 8: 5<sup>th</sup> August - 11<sup>th</sup> August</h1>
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<h2>Plasmid 3:</h2>
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<p>
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05/08/13</br>
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We started the week by doing the golden gates over again, meaning the GG 3 and GG 2 (2.1 with FurBS 1, 2.2 with FurBS2 and 2.3 with FurBS3). In fact, our previous control positive had some unwanted white spots (01/08/13), thus suggesting some contaminations during the transformation step. curretnmy waiting
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</p>
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 +
 
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<p>
 +
06/08/13</br>
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We performed 6 golden gates, 3 for the second construction, 2 for the third construction and one for the controle plasmid.
 +
We prepared a mix for the 6+1 tubes :
 +
  <ul>
 +
    <li> 7x 1,27 = 8,89 µl of 1A3 plasmide
 +
    <li> 7x 1,74 = 12,18 µL of terminator
 +
    <li> 7x 1,5 = 10,5 µL of T4 Buffer
 +
    <li> 7 x 0,5 = 3,5 µL of BSA
 +
    <li> 7 x 0,5 = 3,5 µL of T4 ligase
 +
  </ul>
 +
</p>
 +
<p>
 +
 
 +
We then added:<br>
 +
 
 +
Tube 1. =
 +
  <ul>
 +
    <li>  0,76 µl of Andersen's promoter
 +
    <li>  0,76 µL of RBS
 +
    <li>  0,76 µl OF Fur BS
 +
    <li> 2,93 µL of sfGFP
 +
  </ul>
 +
</p>
 +
 
 +
Tube 2. =
 +
  <ul>
 +
    <li>  0,76 µl of Andersen's promoter
 +
    <li>  0,76 µL of RBS
 +
    <li>  0,76 µl OF Fur BS
 +
    <li> 2,93 µL of sfGFP
 +
  </ul>
 +
</p>
 +
 
 +
Tube 3. =
 +
  <ul>
 +
    <li>  0,76 µl of Andersen's promoter
 +
    <li>  0,76 µL of RBS
 +
    <li>  0,76 µl OF Fur BS
 +
    <li> 2,93 µL of sfGFP
 +
  <li>  4,28 µL of water
 +
  </ul>
 +
</p>
 +
 
 +
Tube 4. =
 +
  <ul>
 +
    <li>  1,25 µl of pLac O
 +
    <li>  µL of RBS-sfGFP
 +
  <li>  3,4 µL of water
 +
  </ul>
 +
</p>
 +
 
 +
Tube 5. =
 +
  <ul>
 +
    <li>  1,25 µl of pLac O
 +
    <li>  2,05 µl of EntA
 +
    <li>  3,37 µL of EntD
 +
    <li>  2,07 µl of EntF
 +
    <li> 0,5 µL of water
 +
  </ul>
 +
</p>
 +
 
 +
Tube 6. = Controle Plasmid
 +
  <ul>
 +
    <li>  0,76 µl of Andersen's promoter
 +
    <li>  0,76 µL of RBS
 +
    <li>  2,93 µl of sfGFP
 +
    <li> 4,28 µL of water
 +
  </ul>
 +
</p>
 +
 
 +
On the afternoon, we launch another golden gate for EntB/C/E
 +
 
 +
Tube 7. =
 +
  <ul>
 +
    <li>  1,27 µl of plasmid 1A3
 +
    <li>  1,5 µL of pLacO
 +
    <li>  0,49 µl of EntB
 +
    <li> 0,65 µL of EntC
 +
    <li>  1,74 µl of terminator
 +
    <li>  1,5 µL of Buffer T4 ligase
 +
    <li>  0,5 µl of T4 ligase
 +
    <li> 0,5 µL of Bsa 1
 +
    <li> 5,88 µL of water
 +
  </ul>
 +
</p>
</br>
</br>
<p>
<p>
-
con</br>
+
Additionnally, we did the optimization of our PCR over with the temperature gradient for the annealing step. current waiting
-
nard</br>
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</p>
</p>
 +
</br>
 +
<p>
 +
09/08
 +
We make 38 tube of Top 10
 +
</p>
 +
 +
</br>
 +
<p>
 +
 +
</p>
 +
 +
</br>
 +
<p>
 +
 +
</p>
 +
 +
</br>
 +
<p>
 +
 +
</p>
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</br>
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<p>
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 +
</p>
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</div>
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</div>
</html>
</html>
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{{:Team:Evry/foot}}

Latest revision as of 07:53, 26 August 2013

Iron coli project

Week 8: 5th August - 11th August

Plasmid 3:

05/08/13
We started the week by doing the golden gates over again, meaning the GG 3 and GG 2 (2.1 with FurBS 1, 2.2 with FurBS2 and 2.3 with FurBS3). In fact, our previous control positive had some unwanted white spots (01/08/13), thus suggesting some contaminations during the transformation step. curretnmy waiting

06/08/13
We performed 6 golden gates, 3 for the second construction, 2 for the third construction and one for the controle plasmid. We prepared a mix for the 6+1 tubes :

  • 7x 1,27 = 8,89 µl of 1A3 plasmide
  • 7x 1,74 = 12,18 µL of terminator
  • 7x 1,5 = 10,5 µL of T4 Buffer
  • 7 x 0,5 = 3,5 µL of BSA
  • 7 x 0,5 = 3,5 µL of T4 ligase

We then added:
Tube 1. =

  • 0,76 µl of Andersen's promoter
  • 0,76 µL of RBS
  • 0,76 µl OF Fur BS
  • 2,93 µL of sfGFP

Tube 2. =
  • 0,76 µl of Andersen's promoter
  • 0,76 µL of RBS
  • 0,76 µl OF Fur BS
  • 2,93 µL of sfGFP

Tube 3. =
  • 0,76 µl of Andersen's promoter
  • 0,76 µL of RBS
  • 0,76 µl OF Fur BS
  • 2,93 µL of sfGFP
  • 4,28 µL of water

Tube 4. =
  • 1,25 µl of pLac O
  • µL of RBS-sfGFP
  • 3,4 µL of water

Tube 5. =
  • 1,25 µl of pLac O
  • 2,05 µl of EntA
  • 3,37 µL of EntD
  • 2,07 µl of EntF
  • 0,5 µL of water

Tube 6. = Controle Plasmid
  • 0,76 µl of Andersen's promoter
  • 0,76 µL of RBS
  • 2,93 µl of sfGFP
  • 4,28 µL of water

On the afternoon, we launch another golden gate for EntB/C/E Tube 7. =
  • 1,27 µl of plasmid 1A3
  • 1,5 µL of pLacO
  • 0,49 µl of EntB
  • 0,65 µL of EntC
  • 1,74 µl of terminator
  • 1,5 µL of Buffer T4 ligase
  • 0,5 µl of T4 ligase
  • 0,5 µL of Bsa 1
  • 5,88 µL of water


Additionnally, we did the optimization of our PCR over with the temperature gradient for the annealing step. current waiting


09/08 We make 38 tube of Top 10





Retrieved from "http://2013.igem.org/Team:Evry/Notebook/w8"