Team:Evry/Notebook/w7

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<h1>Weeks</h1>
 
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<table>
 
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<tr>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w1">1</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w2">2</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w3">3</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w4">4</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w5">5</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w6">6</a></td>
 
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  <td>7</td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w8">8</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w9">9</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w10">10</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w11">11</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w12">12</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w13">13</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w14">14</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w15">15</a></td>
 
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  <td><a href="https://2012.igem.org/Team:Evry/Notebook/w16">16</a></td>
 
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</tr>
 
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</table>
 
<h1>Week 7: 23rd July - 29th July</h1>
<h1>Week 7: 23rd July - 29th July</h1>
-
<h2>Monday, 23rd June</h2>
+
<h2>Monday, 23rd July</h2>
<h3>Cloning:</h3>
<h3>Cloning:</h3>
Line 69: Line 45:
<table/>
<table/>
-
<h2>Tuesday, 24th June</h2>
+
<h3>Xenopus:</h3>
 +
Start of auxin's toxicity test on tadpoles: Tadpoles in there growth media + 0/125/250 or 500 µM auxin<br>
 +
<i>We changed this media each days during one week</i>
 +
<h2>Tuesday, 24th July</h2>
 +
<h3>Plasmid Purification:</h3>
-
<h2>Thursday, 26th June</h2>
+
On the <strong>pCS2+ RFP</strong> clones incubated the day before:
 +
<TABLE BORDER="1">
 +
  <CAPTION> DNA Concentration </CAPTION>
 +
  <TR>
 +
    <TH> <center> Tube </center></TH>
 +
    <TH> <center> Concentration (ng.uL-1) </center></TH>
 +
  </TR>
 +
  <TR>
 +
    <TH> 1 </TH>
 +
    <TD> <center>144,2</center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> 2 </TH>
 +
    <TD> <center>161,9</center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> 3 </TH>
 +
    <TD> <center>184,9</center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> 4 </TH>
 +
    <TD> <center>120,7</center></TD>
 +
  </TR>
 +
</TABLE>
 +
 +
<h3>Digestion:</h3>
 +
 +
<TABLE BORDER="1">
 +
  <CAPTION> Plasmid digestion </CAPTION>
 +
  <TR>
 +
    <TH> <center> Tube </center></TH>
 +
    <TH> <center> V DNA (uL) </center></TH>
 +
    <TH> <center> V SpeI (uL) </center></TH>
 +
    <TH> <center> V EcoRI (uL) </center></TH>
 +
    <TH> <center> V buffer (uL) </center></TH>
 +
    <TH> <center> V H2O (uL) </center></TH>
 +
  </TR>
 +
  <TR>
 +
    <TH> 1 </TH>
 +
    <TD> <center>7</center></TD>
 +
    <TD> <center>1</center></TD>
 +
    <TD> <center>1</center></TD>
 +
    <TD> <center>2</center></TD>
 +
    <TD> <center>9</center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> 2 </TH>
 +
    <TD> <center>6,2</center></TD>
 +
    <TD> <center>1</center></TD>
 +
    <TD> <center>1</center></TD>
 +
    <TD> <center>2</center></TD>
 +
    <TD> <center>9,8</center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> 3 </TH>
 +
    <TD> <center>5,4</center></TD>
 +
    <TD> <center>1</center></TD>
 +
    <TD> <center>1</center></TD>
 +
    <TD> <center>2</center></TD>
 +
    <TD> <center>10,6</center></TD>
 +
  </TR>
 +
  <TR>
 +
    <TH> 4 </TH>
 +
    <TD> <center>8,3</center></TD>
 +
    <TD> <center>1</center></TD>
 +
    <TD> <center>1</center></TD>
 +
    <TD> <center>2</center></TD>
 +
    <TD> <center>7,7</center></TD>
 +
  </TR>
 +
</TABLE>
 +
 +
<h3>Gel migration:</h3>
 +
 +
Gel 0,8%
 +
 +
 +
 +
 +
<h2>Wednesday, 25th July</h2>
 +
 +
Reception of primers fo Auxin Enzymes: IaaH FW and Rv and IaaM FW and RV.<br>
 +
PCR of imperial college BB with these primers to BB IaaH and IaaM.
 +
Electrophoresis show that results aren't good.
 +
 +
<h3>PCR:</h3>
 +
 +
PCR of pCS2+:
 +
 +
<table border="1">
 +
      <tr>
 +
        <td>Reactants</td>
 +
        <td>Volumes (µl)</td>
 +
      </tr>
 +
      <tr>
 +
        <td>GC Buffer</td>
 +
        <td>10</td>
 +
      </tr> 
 +
      <tr>
 +
        <td>dNTPs</td>
 +
        <td>1</td>
 +
      </tr> 
 +
      <tr>
 +
        <td>DNA</td>
 +
        <td>0,5</td>
 +
      </tr> 
 +
      <tr>
 +
        <td>H2O</td>
 +
        <td>33</td>
 +
      </tr> 
 +
      <tr>
 +
        <td>Primers (FW and RV)</td>
 +
        <td>2,5 each</td>
 +
      </tr> 
 +
      <tr>
 +
        <td>fusion polymerase</td>
 +
        <td>0,5</td>
 +
      </tr> 
 +
<table/>
 +
<h3>PCR:</h3>
 +
0,8% agarose
 +
<h2>Thursday, 26th July</h2>
-
<h2>Friday, 27th June</h2>
+
Test of auxin toxicity in tadpodes.
 +
Retry of IaaH and IaaM BB: Ok for IaaM but not for IaaH.
 +
<h2>Friday, 27th July</h2>
 +
<ul>
 +
<li>PCR of TirI: One PCR with primers TirI FW + Sdm RV / One PCR with primers TirI RV + Sdm FW
 +
<li>Gel extraction of these PCR
 +
<li>New PCR with the mix of the two PCR products + primers TirI FW and RV
 +
</ul>
</html>
</html>

Latest revision as of 10:01, 9 August 2012

Weeks:

June July August September October November

Week 7: 23rd July - 29th July

Monday, 23rd July

Cloning:

4 different clones pCS2+ RFP => incubation in LB medium overnight at 37 degree celsius.

PCR:

PCR of pCS2+:
Reactants Volumes (µl)
GC Buffer 10
dNTPs 1
DNA 1
H2O 32,5
Primers (FW and RV) 2,5 each
fusion polymerase 0,5

Xenopus:

Start of auxin's toxicity test on tadpoles: Tadpoles in there growth media + 0/125/250 or 500 µM auxin
We changed this media each days during one week

Tuesday, 24th July

Plasmid Purification:

On the pCS2+ RFP clones incubated the day before:
DNA Concentration
Tube
Concentration (ng.uL-1)
1
144,2
2
161,9
3
184,9
4
120,7

Digestion:

Plasmid digestion
Tube
V DNA (uL)
V SpeI (uL)
V EcoRI (uL)
V buffer (uL)
V H2O (uL)
1
7
1
1
2
9
2
6,2
1
1
2
9,8
3
5,4
1
1
2
10,6
4
8,3
1
1
2
7,7

Gel migration:

Gel 0,8%

Wednesday, 25th July

Reception of primers fo Auxin Enzymes: IaaH FW and Rv and IaaM FW and RV.
PCR of imperial college BB with these primers to BB IaaH and IaaM. Electrophoresis show that results aren't good.

PCR:

PCR of pCS2+:
Reactants Volumes (µl)
GC Buffer 10
dNTPs 1
DNA 0,5
H2O 33
Primers (FW and RV) 2,5 each
fusion polymerase 0,5

PCR:

0,8% agarose

Thursday, 26th July

Test of auxin toxicity in tadpodes. Retry of IaaH and IaaM BB: Ok for IaaM but not for IaaH.

Friday, 27th July

  • PCR of TirI: One PCR with primers TirI FW + Sdm RV / One PCR with primers TirI RV + Sdm FW
  • Gel extraction of these PCR
  • New PCR with the mix of the two PCR products + primers TirI FW and RV

Retrieved from "http://2012.igem.org/Team:Evry/Notebook/w7"