Difference between revisions of "Team:Pasteur Paris/Week 12"

Line 101: Line 101:
 
               <span class="soustitrepage">08/17 - 08/21</span></p>
 
               <span class="soustitrepage">08/17 - 08/21</span></p>
 
               <p></p><br><br><br>
 
               <p></p><br><br><br>
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               <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><em>Gibson  Assembly</em></p><br />
 +
<ul>
 +
<li>Gel  Migration of the PCR products (Cluster 2 and BBa_808014)</li>
 +
<li>PCR  Amplification of intersequence 936011/936023 using Takara Ex Taq Polymerase.</li>
 +
<li>Gel  Migration on 0,8% Agarose Gel</li>
 +
<li>PCR  Amplification of BBa_316003 using Takara Ex Taq Polymerase.</li>
 +
<li>Gel  Migration using 0,8% Agarose Gel</li>
 +
<li>Spe I and  Pst I Restriction Digest of the recieved plasmids containing BBa_K936011, BBa_K13932932, K936023.</li>
 +
<li>Gel Purification of the digested  plasmids K936011 and K1392932 using the QIAgen Gel extraction kit protocol. </li>
 +
</ul>
  
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+
<p><em>NB  -Esterase assay </em></p>  
  
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+
<ul>
 +
<li>Liquid culture of  the second transformation (of the 15/08/15): BBa_808030 and pDG011 plasmid in DH5α </li>
 +
<li>MiniPrep of  BBa_808030 in the plasmid pDG011 in BAP 1</li>
 +
<li>XbaI and PstI digestion  of BBa_808030 and pDG011 (with the phosphatase step)</li>
 +
</ul>
  
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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/>
+
<p><em>Gel Purification</em></p>
 +
 
 +
<p><u>Tubes components:</u></p>
 +
<table border="1" cellspacing="0" cellpadding="0" width="480">
 +
  <tr>
 +
    <td width="102" valign="top"><br />
 +
      Tubes </td>
 +
    <td width="217" valign="top"><p align="center">Ladder </p></td>
 +
    <td width="161" valign="top"><p align="center">1st transformation </p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="102" valign="top"><p>&nbsp;</p>
 +
      <p>Components </p></td>
 +
    <td width="217" valign="top"><p align="center"><strong>&nbsp;</strong></p>
 +
      <p align="center"><strong>GeneRuler 1kb DNA Ladder</strong><br />
 +
        (6x DNA    loading dye)<br />
 +
        3µL</p></td>
 +
    <td width="161" valign="top"><p align="center"><strong>Buffer</strong><br />
 +
      4µL<br />
 +
      +<br />
 +
      <strong>DNA</strong> <br />
 +
      20µL</p></td>
 +
  </tr>
 +
</table>
 +
 
 +
<ul>
 +
  <li>Ligation of BBa_808030 and pDG011 </li>
 +
  <li>Transformation in Chemically Competent DH5-⍺ cells </li>
 +
 
 +
<li>Transformation  in DH5-ɑ of the biobrick K808030 in pDG011 </li>
 +
<li>Transformation in DH5α cells of BBa_808030 insert and pDG011 vector </li>
 +
 
 +
 
 +
  <li>SpeI and Pst  I Enzymatic Digest of BBa_K808030 with dephosphorylation.</li>
 +
  <li>Gel migration</li>
 +
  <li>Gel  extraction of the PCR product of K808030 and pDG011.</li>
 +
  <li>Ligation of  pDG011 and K808030.</li>
 +
</ul>
 +
 
 +
<p>&nbsp;</p>
 +
<p>The bacteria did not grow  because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not  pSB1C3.<br />
 +
  - Bacterial culture  of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well. <br />
 +
  - Midi-prep has been  done with the 50mL of BBa_J61047 culture. (dilution 1/1000) </p>
 +
<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">0,5 </p></td>
 +
    <td width="121" valign="top"><p align="right">0,011 </p></td>
 +
    <td width="121" valign="top"><p align="right">0,004 </p></td>
 +
    <td width="121" valign="top"><p align="right">2,57 </p></td>
 +
  </tr>
 +
</table>
 +
<ul> <li>Preparation of YPD  specific media on specific media (1%  yeast extract, 2% Dextrose, 2% Peptone, 2% agar).</li>
 +
 
 +
  <li>Gel Migration of BBa_J61047.</li>
 +
  <li>NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme. </li>
 +
  <li>Gel Migration on a 0,8%  Agarose Gel and the digestion hasn&rsquo;t work. </li>
 +
  <li>XbaI and SpeI digestion of BBa_J61047. </li>
 +
  </ul>
 +
 
 +
  <ul>
 +
    <li>tube 1 «&nbsp;Miniprep XbaI/SpeI&nbsp;»&nbsp;:&nbsp;5uL 10X buffer cutsmart + 0,5uL  XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.</li>
 +
    <li>tube 2 «&nbsp;Midiprep XbaI/SpeI&nbsp;»&nbsp;: 5uL 10X buffer cut smart + 0,5uL XbaI +  0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 +
    <li>tube 3 «&nbsp;Miniprep NotI&nbsp;»&nbsp;: 5uL 10X buffer cutsmart + 0,5 NotI +  38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 +
    <li>tube 4 «&nbsp;MiDiprep NotI&nbsp;»&nbsp;: 5uL 10X buffer cutsmart + 0,5 NotI +  38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 +
    <li>tube 5 «&nbsp;Miniprep CTRL&nbsp;» as negative  control&nbsp;: 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.</li>
 +
    <li>tube 6 «&nbsp;Midiprep CTRL&nbsp;» as negative  control&nbsp;: 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.<li>
 +
    <li>MWT&nbsp;: «&nbsp;Quick-Load Purple 2-Log DNA  Ladder (0.1-10.0 kb) NEB </li>
 +
</ul>
 +
 
 +
</br>
 +
 
 +
<ul>
 +
  <li>PCR Amplification of the  BBa_J61047 using Phusion Polymerase Master Mix. </li>
 +
  <li>Gel Migration on a 0.8% agarose gel (See figure 01)<br />
 +
  </li>
 +
  <li>Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h. </li>
 +
  <li>PCR Amplification using the Phusion Polymerase of pRS415. </li>
 +
</ul>
 +
 
 +
<p><em>NB-Esterase Assay: </em></p>
 +
<ul>
 +
<li>MiniPrep 808030 in the plasmid pDG011</li>
 +
<li>Gibson Assembly</li>
 +
<li>OD measurement of Cluster 4</li>
 +
</ul>
 +
 
 +
<div align="center">
 +
  <table border="1" cellspacing="0" cellpadding="0" width="651">
 +
    <tr>
 +
      <td width="121" valign="top"><p>&nbsp;</p></td>
 +
      <td width="99" valign="top"><p align="center">Concentration (ng/µL) </p></td>
 +
      <td width="83" valign="top"><p align="center">OD(230nm) </p></td>
 +
      <td width="83" valign="top"><p align="center">OD(260nm) </p></td>
 +
      <td width="83" valign="top"><p align="center">OD(280nm) </p></td>
 +
      <td width="91" valign="top"><p align="center">OD(260nm)/OD(230nm) </p></td>
 +
      <td width="91" valign="top"><p align="center">OD(260nm)/OD(280nm) </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="121" valign="top"><p align="center">Cluster_4_start </p></td>
 +
      <td width="99" valign="top"><p align="center">30 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,025 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,575 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,375 </p></td>
 +
      <td width="91" valign="top"><p align="center">1,50 </p></td>
 +
      <td width="91" valign="top"><p align="center">- </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="121" valign="top"><p align="center">/936011 </p></td>
 +
      <td width="99" valign="top"><p align="center">55 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,4 </p></td>
 +
      <td width="83" valign="top"><p align="center">1,075 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,575 </p></td>
 +
      <td width="91" valign="top"><p align="center">1,88 </p></td>
 +
      <td width="91" valign="top"><p align="center">2,65 </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="121" valign="top"><p align="center">936011/936023 </p></td>
 +
      <td width="99" valign="top"><p align="center">7,5 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,6 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,125 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,05 </p></td>
 +
      <td width="91" valign="top"><p align="center">2,41 </p></td>
 +
      <td width="91" valign="top"><p align="center">0,21 </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="121" valign="top"><p align="center">936023/ </p></td>
 +
      <td width="99" valign="top"><p align="center">45 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,575 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,925 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,625 </p></td>
 +
      <td width="91" valign="top"><p align="center">1,48 </p></td>
 +
      <td width="91" valign="top"><p align="center">1,61 </p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="121" valign="top"><p align="center">Cluster_4_end </p></td>
 +
      <td width="99" valign="top"><p align="center">47,5 </p></td>
 +
      <td width="83" valign="top"><p align="center">0 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,925 </p></td>
 +
      <td width="83" valign="top"><p align="center">0,575 </p></td>
 +
      <td width="91" valign="top"><p align="center">1,64 </p></td>
 +
      <td width="91" valign="top"><p align="center">- </p></td>
 +
    </tr>
 +
  </table>
 +
</div>
  
−
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             </td>
 
             </td>

Revision as of 21:03, 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 12

08/17 - 08/21




Gibson Assembly


  • Gel Migration of the PCR products (Cluster 2 and BBa_808014)
  • PCR Amplification of intersequence 936011/936023 using Takara Ex Taq Polymerase.
  • Gel Migration on 0,8% Agarose Gel
  • PCR Amplification of BBa_316003 using Takara Ex Taq Polymerase.
  • Gel Migration using 0,8% Agarose Gel
  • Spe I and Pst I Restriction Digest of the recieved plasmids containing BBa_K936011, BBa_K13932932, K936023.
  • Gel Purification of the digested plasmids K936011 and K1392932 using the QIAgen Gel extraction kit protocol.

NB -Esterase assay

  • Liquid culture of the second transformation (of the 15/08/15): BBa_808030 and pDG011 plasmid in DH5α
  • MiniPrep of BBa_808030 in the plasmid pDG011 in BAP 1
  • XbaI and PstI digestion of BBa_808030 and pDG011 (with the phosphatase step)

Gel Purification

Tubes components:


Tubes

Ladder

1st transformation

 

Components

 

GeneRuler 1kb DNA Ladder
(6x DNA loading dye)
3µL

Buffer
4µL
+
DNA
20µL
  • Ligation of BBa_808030 and pDG011
  • Transformation in Chemically Competent DH5-⍺ cells
  • Transformation in DH5-ɑ of the biobrick K808030 in pDG011
  • Transformation in DH5α cells of BBa_808030 insert and pDG011 vector
  • SpeI and Pst I Enzymatic Digest of BBa_K808030 with dephosphorylation.
  • Gel migration
  • Gel extraction of the PCR product of K808030 and pDG011.
  • Ligation of pDG011 and K808030.

 

The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.
- Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.
- Midi-prep has been done with the 50mL of BBa_J61047 culture. (dilution 1/1000)

 

DNA Concentration (ng/µl)

OD(260nm)

OD(280nm)

OD(260nm)/OD(280nm)

BBa_J61047

0,5

0,011

0,004

2,57

  • Preparation of YPD specific media on specific media (1% yeast extract, 2% Dextrose, 2% Peptone, 2% agar).
  • Gel Migration of BBa_J61047.
  • NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme.
  • Gel Migration on a 0,8% Agarose Gel and the digestion hasn’t work.
  • XbaI and SpeI digestion of BBa_J61047.
  • tube 1 « Miniprep XbaI/SpeI » : 5uL 10X buffer cutsmart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.
  • tube 2 « Midiprep XbaI/SpeI » : 5uL 10X buffer cut smart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour
  • tube 3 « Miniprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
  • tube 4 « MiDiprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
  • tube 5 « Miniprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
  • tube 6 « Midiprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
  • MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB

  • PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.
  • Gel Migration on a 0.8% agarose gel (See figure 01)
  • Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h.
  • PCR Amplification using the Phusion Polymerase of pRS415.

NB-Esterase Assay:

  • MiniPrep 808030 in the plasmid pDG011
  • Gibson Assembly
  • OD measurement of Cluster 4

 

Concentration (ng/µL)

OD(230nm)

OD(260nm)

OD(280nm)

OD(260nm)/OD(230nm)

OD(260nm)/OD(280nm)

Cluster_4_start

30

0,025

0,575

0,375

1,50

-

/936011

55

0,4

1,075

0,575

1,88

2,65

936011/936023

7,5

0,6

0,125

0,05

2,41

0,21

936023/

45

0,575

0,925

0,625

1,48

1,61

Cluster_4_end

47,5

0

0,925

0,575

1,64

-

^
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