Difference between revisions of "Team:Pasteur Paris/Week 13"

 
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{{Pasteur_Paris}}
 
{{Pasteur_Paris}}
 
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  <center>
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        <tr  vertical-align:top;>
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            <td class="menunotebook" style="margin: 0px; vertical-align: top;">
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              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_1" class="week">
 +
                  <h1>Week&nbsp;1</h1>
 +
              </a>
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              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_2" class="week">
 +
                  <h1>Week&nbsp;2</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_3" class="week">
 +
                  <h1>Week&nbsp;3</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_4" class="week">
 +
                  <h1>Week&nbsp;4</h1>
 +
              </a>
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              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_5" class="week">
 +
                  <h1>Week&nbsp;5</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_6" class="week">
 +
                  <h1>Week&nbsp;6</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_7" class="week">
 +
                  <h1>Week&nbsp;7</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_8" class="week">
 +
                  <h1>Week&nbsp;8</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_9" class="week">
 +
                  <h1>Week&nbsp;9</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_10" class="week">
 +
                  <h1>Week&nbsp;10</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_11" class="week">
 +
                  <h1>Week&nbsp;11</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_12" class="week">
 +
                  <h1>Week&nbsp;12</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_13" class="week">
 +
                  <h1>Week&nbsp;13</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_14" class="week">
 +
                  <h1>Week&nbsp;14</h1>
 +
              </a>
 +
              <a href="https://2015.igem.org/Team:Pasteur_Paris/Week_15" class="week">
 +
                  <h1>Week&nbsp;15</h1>
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              </a>
 +
            </td>
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            <td style="vertical-align: top; width: 90%;" class="fontcorpspage">
 +
              <p><span class="titrepage">Week&nbsp;13</span><br/><br/>
 +
              <span class="soustitrepage">08/24 - 08/28</span></p>
 +
                <p></p><br>
 +
 +
<center><h3><em>Yeast assembly</em></h3></center></br>
 +
<ul style="text-align: left">
 +
<li>Treatment of the yeast using a homemade Zymolyase solution&nbsp;:</li>
 +
<ol>
 +
  <li> PBS pH 7.5 5 ml</li>
 +
  <li>glucose 5% to make a 2%  solution</li>
 +
  <li>Zymolyase 0.1 g (0.012-0.022 mg/mL)</li>
 +
  <li>Distilled water free RNAs/DNAs 1 mL</li>
 +
  <li>MidiPrep of pRS415</li>
 +
</ol>
 +
</ul>
 +
 +
<table border="1" cellspacing="0" cellpadding="0" width="604">
 +
  <tr>
 +
    <td width="86" valign="top"><br />
 +
      Dilution 1/50 </td>
 +
    <td width="86" valign="top"><p>DNA Concentration (ng/µl)</p></td>
 +
    <td width="86" valign="top"><p>OD(230nm)</p></td>
 +
    <td width="86" valign="top"><p>OD(260nm)</p></td>
 +
    <td width="86" valign="top"><p>OD(280nm)</p></td>
 +
    <td width="86" valign="top"><p>OD(260nm)/OD(230nm)</p></td>
 +
    <td width="86" valign="top"><p>OD(260nm)/OD(280nm)</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="86" valign="top"><p>pRS415</p></td>
 +
    <td width="86" valign="top"><p align="right">6.2 </p></td>
 +
    <td width="86" valign="top"><p align="right">0.122 </p></td>
 +
    <td width="86" valign="top"><p align="right">0.067 </p></td>
 +
    <td width="86" valign="top"><p align="right">0.076 </p></td>
 +
    <td width="86" valign="top"><p align="right">1.60 </p></td>
 +
    <td width="86" valign="top"><p align="right">1.83 </p></td>
 +
  </tr>
 +
</table>
 +
 +
<p style="text-align: left">MidiPrep has been  digested to verified if the DNA correspond to the plasmid of interest or to an  other plasmid that is naturally present in  yeast. </p>
 +
<ul style="text-align: left">
 +
  <li>BamHI digestion of pRS415  (with the phosphatase step). </li>
 +
</ul>
 +
<p style="text-align: left">We can see the presence of a streak about  7500 bp which seems to correspond to our plasmid pRS415.</p>
 +
<ul style="text-align: left">
 +
  <li>DNA concentration  measurement. </li>
 +
</ul>
 +
<table border="1" cellspacing="0" cellpadding="0" width="604">
 +
  <tr>
 +
    <td width="121" valign="top"><p>&nbsp;</p></td>
 +
    <td width="121" valign="top"><p>DNA Concentration (ng/µl)</p></td>
 +
    <td width="121" valign="top"><p>OD(260nm)</p></td>
 +
    <td width="121" valign="top"><p>OD(280nm)</p></td>
 +
    <td width="121" valign="top"><p>OD(260nm)/OD(280nm)</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="121" valign="top"><p>BBa_J61047</p></td>
 +
    <td width="121" valign="top"><p align="right">0.4 </p></td>
 +
    <td width="121" valign="top"><p align="right">0.008 </p></td>
 +
    <td width="121" valign="top"><p align="right">0.002 </p></td>
 +
    <td width="121" valign="top"><p align="right">3.76 </p></td>
 +
  </tr>
 +
</table>
 +
 +
<ul style="text-align: left">
 +
  <li>PCR amplification of pRS415  with a mutagenesis kit using TE 8.1. </li>
 +
  <li>PCR amplification of pRS415  with a mutagenesis kit to insert nicks without TE 8.1.<br />
 +
    A second trial has been done and TE 8.1 has been replaced by DNAse RNAse free water. </li>
 +
  <li>Gel migration on a 0.7%  agarose gel. </li>
 +
 +
  <li>PCR amplification of pRS415  with new recommandations (protocol n°3) </li>
 +
  <li>Gel migration on a 0.7%  agarose gel.<br />
 +
  </li>
 +
 +
  <li>Preparation of  electro-competent yeast cells.</li>
 +
  <li>Electroporation of  pRS415 and Cre with overlapping sequences. </li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<center><h3><em>TPA solubility</em></h3></center></br>
 +
<ul style="text-align: left">
 +
  <li>improvement of TPA  solubility in different conditions : </li>
 +
  <ol>
 +
          <li>TPA (0.1 g) + Na<sub>2</sub>H<sub>2</sub>PO<sub>4</sub>(0.5 g)  in 150 ml of water: complete TPA solubilization. </li>
 +
          <li>TPA (0.1 g) + NaOH(0.5 g) in  150 ml of water: complete TPA solubilization. </li>
 +
  </ol>
 +
  <li>Fluorescence measurements : </li>
 +
  <ol>
 +
          <li>no TPAOH was observed on our  measurements, the reaction worked. </li>
 +
  </ol>
 +
</ul>
 +
<br>
 
<br>
 
<br>
−
<p> week 13 </p>
+
<a class="hautdepage" href="#"><p><span style="font-size: 60px; font-family: Courier;">^</span><br/><span style="font-size: 20px;">Page up</span></p></a></p>
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            </td>
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        </tr>
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Latest revision as of 23:47, 3 October 2015

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 13

08/24 - 08/28


Yeast assembly


  • Treatment of the yeast using a homemade Zymolyase solution :
    1. PBS pH 7.5 5 ml
    2. glucose 5% to make a 2% solution
    3. Zymolyase 0.1 g (0.012-0.022 mg/mL)
    4. Distilled water free RNAs/DNAs 1 mL
    5. MidiPrep of pRS415

Dilution 1/50

DNA Concentration (ng/µl)

OD(230nm)

OD(260nm)

OD(280nm)

OD(260nm)/OD(230nm)

OD(260nm)/OD(280nm)

pRS415

6.2

0.122

0.067

0.076

1.60

1.83

MidiPrep has been digested to verified if the DNA correspond to the plasmid of interest or to an other plasmid that is naturally present in yeast.

  • BamHI digestion of pRS415 (with the phosphatase step).

We can see the presence of a streak about 7500 bp which seems to correspond to our plasmid pRS415.

  • DNA concentration measurement.

 

DNA Concentration (ng/µl)

OD(260nm)

OD(280nm)

OD(260nm)/OD(280nm)

BBa_J61047

0.4

0.008

0.002

3.76

  • PCR amplification of pRS415 with a mutagenesis kit using TE 8.1.
  • PCR amplification of pRS415 with a mutagenesis kit to insert nicks without TE 8.1.
    A second trial has been done and TE 8.1 has been replaced by DNAse RNAse free water.
  • Gel migration on a 0.7% agarose gel.
  • PCR amplification of pRS415 with new recommandations (protocol n°3)
  • Gel migration on a 0.7% agarose gel.
  • Preparation of electro-competent yeast cells.
  • Electroporation of pRS415 and Cre with overlapping sequences.

 

TPA solubility


  • improvement of TPA solubility in different conditions :
    1. TPA (0.1 g) + Na2H2PO4(0.5 g) in 150 ml of water: complete TPA solubilization.
    2. TPA (0.1 g) + NaOH(0.5 g) in 150 ml of water: complete TPA solubilization.
  • Fluorescence measurements :
    1. no TPAOH was observed on our measurements, the reaction worked.


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