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

Line 102: Line 102:
 
               <span class="soustitrepage">08/24 - 08/28</span></p>
 
               <span class="soustitrepage">08/24 - 08/28</span></p>
 
                 <p></p><br><br><br>
 
                 <p></p><br><br><br>
−
               <p style="text-align: justify; padding: 30px;">Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec et purus vel risus iaculis tristique. Ut sit amet eros mi. Nulla elementum aliquam nibh. Praesent commodo, leo sit amet luctus gravida, dolor orci fermentum dolor, et consequat velit lacus quis ligula. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Fusce ut euismod odio. Vestibulum sed congue velit. Donec vel mi eget felis elementum interdum quis ac nisi.<br/><br/>
+
               <p style="text-align: left"
 +
<h3>Yeast Assembly</h3>
 +
<ul>
 +
<li>Treatment of the yeast using a homemade Zymolyase solution&nbsp;:</li>
 +
<ol>
 +
  <li>pH7,5 PBS 5mL</li>
 +
  <li>glucose 5% to make a 2%  solution</li>
 +
  <li>Zymolyase 0,1g (0,012-0,022mg/mL)</li>
 +
  <li>Distilled water free RNAs/DNAs 1mL</li>
 +
  <li>MidiPrep of pRS415</li>
 +
</ol>
 +
</ul>
  
−
Suspendisse ultricies tortor et elementum aliquam. Sed eu arcu nisi. Sed efficitur semper lacinia. Integer egestas mi id metus aliquam, id porta nisl finibus. Maecenas lobortis felis nec laoreet interdum. Sed tristique accumsan tincidunt. Suspendisse sit amet lacus id massa ultrices luctus quis a urna. Duis euismod turpis vitae urna dictum dictum. Sed ut lacinia augue, interdum euismod risus. Vivamus finibus laoreet quam, quis fringilla elit porta vel. Sed ullamcorper sagittis ante at commodo.<br/><br/>
+
<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>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>
 +
  <li>BamHI digestion of pRS415  (with the phosphatase step). </li>
 +
</ul>
 +
<p align="center">We can see the presence of a streak about  7500bp wich seems to correspond to our plasmid pRS415.</p>
 +
<ul>
 +
  <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>
 +
  <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>
  
−
Quisque et consectetur nisi. In eget iaculis nisi. Duis lacinia, ex eu mollis ornare, velit nisl vestibulum mi, at malesuada velit mi in lorem. Suspendisse sollicitudin sapien sit amet lacus pellentesque, non ultrices quam consectetur. Morbi sapien quam, faucibus vitae rhoncus et, volutpat sed metus. Integer odio neque, imperdiet eu luctus at, molestie eget ligula. Morbi vel dui quis erat venenatis iaculis. Vestibulum pulvinar, quam ac tempor efficitur, odio odio congue lectus, quis euismod orci mi a massa.<br/><br/>
+
  <li>PCR amplification of pRS415  with new recommandations (protocol n°3) </li>
 +
  <li>Gel migration on a 0.7%  agarose gel.<br />
 +
  </li>
  
−
Quisque scelerisque sollicitudin convallis. In non semper dui. Proin eu dignissim justo. Suspendisse dictum imperdiet ante quis rhoncus. Nunc mollis tempor orci eget elementum. Morbi sit amet massa eleifend, egestas lectus nec, condimentum ligula. Pellentesque sit amet scelerisque ipsum.<br/><br/>
+
  <li>Preparation of  electro-competent yeast cells.</li>
 +
  <li>Electroporation of  pRS415 and Cre with overlapping sequences. </li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<h3>TPA solubility : </h3>
 +
<ul>
 +
  <li>improvement of TPA  solubility in different conditions : </li>
 +
  <ol>
 +
        <ol>
 +
          <li>TPA (0.1g) + Na2H2PO4(0.5g)  in 150ml of water: complete TPA solubilization. </li>
 +
          <li>TPA (0.1g) + NaOH(0.5g) in  150ml of water: complete TPA solubilization. </li>
 +
        </ol>
 +
  </ol>
 +
  <li>Fluorescence measurements : </li>
 +
  <ol>
 +
          <li>no TPAOH was observed on our  measurements, the reaction worked. </li>
 +
  </ol>
 +
</ul>
  
−
Quisque posuere ex sed tortor pulvinar finibus dignissim ac lorem. Vivamus quis ullamcorper odio. Mauris ut semper mi. Phasellus metus nisi, tempor id dolor ac, condimentum scelerisque odio. Vivamus ultricies pellentesque metus. Etiam nec metus felis. Nullam facilisis sit amet ante gravida laoreet. Etiam vulputate commodo semper. Curabitur a rutrum est, id bibendum sem. Sed congue semper mauris, sed laoreet erat pulvinar non.<br/><br/>
 
 
<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>
 
<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>
 
             </td>
 
             </td>

Revision as of 22:35, 18 September 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. pH7,5 PBS 5mL
    2. glucose 5% to make a 2% solution
    3. Zymolyase 0,1g (0,012-0,022mg/mL)
    4. Distilled water free RNAs/DNAs 1mL
    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 7500bp wich 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 (0.1g) + Na2H2PO4(0.5g) in 150ml of water: complete TPA solubilization.
      2. TPA (0.1g) + NaOH(0.5g) in 150ml of water: complete TPA solubilization.
  • Fluorescence measurements :
    1. no TPAOH was observed on our measurements, the reaction worked.

^
Page up