Team:UNIPV-Pavia/Calendar/July/settimana4

From 2011.igem.org

(Difference between revisions)
 
(29 intermediate revisions not shown)
Line 12: Line 12:
</p>
</p>
<a name="July.2C_18th"></a><h2> <span class="mw-headline">July, 18th</span></h2>
<a name="July.2C_18th"></a><h2> <span class="mw-headline">July, 18th</span></h2>
 +
<p>
<p>
 +
<a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> in <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61002">BBa_J61002</a> plasmid purification and quantification was carried out:
 +
</p>
 +
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Plasmid</b></td>
 +
      <td class="row"><b>DNA (ng/&mu;l)</b></td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> in <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61002">BBa_J61002</a></td>
 +
      <td class="row">37.3</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
<p>
<p>
Line 19: Line 37:
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Plasmid</b></td>
+
       <td class="row"><b>Plasmid</b></td>
-
       <td><b>Kind</b></td>
+
       <td class="row"><b>Kind</b></td>
-
       <td><b>DNA (&mu;l)</b></td>
+
       <td class="row"><b>DNA (&mu;l)</b></td>
-
       <td><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
+
       <td class="row"><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
-
       <td><b>Enzyme 1 (&mu;l)</b></td>
+
       <td class="row"><b>Enzyme 1 (&mu;l)</b></td>
-
       <td><b>Enzyme 2 (&mu;l)</b></td>
+
       <td class="row"><b>Enzyme 2 (&mu;l)</b></td>
-
       <td><b>Buffer H (&mu;l)</b></td>
+
       <td class="row"><b>Buffer H (&mu;l)</b></td>
-
       <td><b>Final Volume (&mu;l)</b></td>
+
       <td class="row"><b>Final Volume (&mu;l)</b></td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E9-2<html></td>
+
       <td class="row">E9-2</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>11.5</td>
+
       <td class="row">11.5</td>
-
       <td>9</td>
+
       <td class="row">9</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 Xbal</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E10-1<html></td>
+
       <td class="row">E10-1</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>10</td>
+
       <td class="row">10</td>
-
       <td>10.5</td>
+
       <td class="row">10.5</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 Xbal</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E11-1<html></td>
+
       <td class="row">E11-1</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>4</td>
+
       <td class="row">4</td>
-
       <td>16.5</td>
+
       <td class="row">16.5</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 Xbal</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E12-1<html></td>
+
       <td class="row">E12-1</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>10</td>
+
       <td class="row">10</td>
-
       <td>10.5</td>
+
       <td class="row">10.5</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 Xbal</td>
-
       <td>1 PstI</td>
+
       <td class="row">1 PstI</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E13-1<html></td>
+
       <td class="row">E13-1</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>6</td>
+
       <td class="row">6</td>
-
       <td>14.5</td>
+
       <td class="row">14.5</td>
-
       <td>1 EcoRl</td>
+
       <td class="row">1 EcoRl</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E16-1<html></td>
+
       <td class="row">E16-1</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>4</td>
+
       <td class="row">4</td>
-
       <td>16.5</td>
+
       <td class="row">16.5</td>
-
       <td>1 EcoRl</td>
+
       <td class="row">1 EcoRl</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E21-1<html></td>
+
       <td class="row">E21-1</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>7.5</td>
+
       <td class="row">7.5</td>
-
       <td>13</td>
+
       <td class="row">13</td>
-
       <td>1 EcoRl</td>
+
       <td class="row">1 EcoRl</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E22-2<html></td>
+
       <td class="row">E22-2</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>10</td>
+
       <td class="row">10</td>
-
       <td>10.5</td>
+
       <td class="row">10.5</td>
-
       <td>1 EcoRl</td>
+
       <td class="row">1 EcoRl</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html><partinfo>BBa_I13521</partinfo><html></td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13521">BBa_I13521</a></td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>12.5</td>
+
       <td class="row">12.5</td>
-
       <td>8</td>
+
       <td class="row">8</td>
-
       <td>1 EcoRl</td>
+
       <td class="row">1 EcoRl</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html><partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo><html></td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> in <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61002">BBa_J61002</a></td>
-
       <td>Vector</td>
+
       <td class="row">Vector</td>
-
       <td>20.5</td>
+
       <td class="row">20.5</td>
-
       <td>0</td>
+
       <td class="row">0</td>
-
       <td>1 EcoRl</td>
+
       <td class="row">1 EcoRl</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
Line 152: Line 170:
<p>
<p>
-
Reactions were incubated at 37°C for three hours; meanwhile two medium-size agarose gels were prepared according to protocols. Moreover <partinfo>BBa_I13521</partinfo> plasmid purification and quantification were carried out:
+
Reactions were incubated at 37°C for three hours; meanwhile two medium-size agarose gels were prepared according to protocols.
-
</p>
+
<br>
 +
According to protocols, 120 &mu;l of 1kb Plus DNA ladder were prepared.
<p>
<p>
Line 159: Line 178:
</p>
</p>
-
<center>
+
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_18_07_2011_E9-XP_E10-XP_E11-XP_E12-XP_E13-EP_E16-EP_E21-EP.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/3/39/UNIPV_18_07_2011_E9-XP_E10-XP_E11-XP_E12-XP_E13-EP_E16-EP_E21-EP.png" class="thumbimage" height="80%" width="80%"></a> <div class="thumbcaption">Medium size gel</div></div></div></div>
-
<table border="1">
+
-
    <tr>
+
-
      <td><b>Plasmid</b></td>
+
-
      <td><b>DNA (ng/&mu;l)</b></td>
+
-
  </tr>
+
-
  <tr>
+
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_18_07_2011_E22_E-P_E23_E-P_I13251_E-P_R0040J61002_E-P.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f8/UNIPV_18_07_2011_E22_E-P_E23_E-P_I13251_E-P_R0040J61002_E-P.png" class="thumbimage" height="80%" width="80%"></a> <div class="thumbcaption">Medium size gel</div></div></div></div>
-
      <td></html><partinfo>BBa_I13521</partinfo><html></td>
+
-
      <td>37.3</td>
+
-
  </tr>
+
-
</table>
 
-
</center>
 
-
 
-
</html>
 
-
 
-
<html>
 
-
<p>2 righe sopra inserire l' immagine</p>
 
<p>
<p>
-
As shown in figure, all clones were positive (apart from E13 run where it is possible to recognize only a band at approximately 2800 bp, corresponding to the undigested plasmid), so we cut and purified the bands of interest. Cells harbouring E13 were again inoculated in 5 ml LB + Amp.
+
As shown in figure, all clones were positive (apart from E13-1 run where it is possible to recognize only a band at approximately 2800 bp, corresponding to the linearized plasmid), so we cut and purified the bands of interest.
</p>
</p>
<p>
<p>
-
E13 was newly then digested with restriction endonucleases:
+
After gel extraction, digested DNA was quantified:
</p>
</p>
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Plasmid</b></td>
+
       <td class="row"><b>Part</b></td>
-
      <td><b>Kind</b></td>
+
       <td class="row"><b>DNA (ng/&mu;l)</b></td>
-
       <td><b>DNA (&mu;l)</b></td>
+
-
      <td><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
+
-
      <td><b>Enzyme 1 (&mu;l)</b></td>
+
-
      <td><b>Enzyme 2 (&mu;l)</b></td>
+
-
      <td><b>Buffer H (&mu;l)</b></td>
+
-
      <td><b>Final Volume (&mu;l)</b></td>
+
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html><partinfo>BBa_I13521</partinfo><html></td>
+
       <td class="row">E9-2 (X-P)</td>
-
       <td>Insert</td>
+
       <td class="row">5.9</td>
-
      <td>1</td>
+
-
      <td>19.5</td>
+
-
      <td>1 EcoRl</td>
+
-
      <td>1 Pstl</td>
+
-
      <td>2.5</td>
+
-
      <td>25</td>
+
   </tr>
   </tr>
-
</table>
 
-
</center>
 
-
 
-
<p>
 
-
After gel extraction, cut DNA was quantified:
 
-
</p>
 
-
 
-
 
-
<center>
 
-
<table border="1">
 
-
    <tr>
 
-
      <td><b>Plasmid</b></td>
 
-
      <td><b>DNA (ng/&mu;l)</b></td>
 
-
  </tr>
 
   <tr>
   <tr>
-
       <td>E9 (X-P)</td>
+
       <td class="row">E10-1 (X-P)</td>
-
       <td>5.9</td>
+
       <td class="row">4.2</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E10 (X-P)</td>
+
       <td class="row">E11-1 (X-P)</td>
-
       <td>4.2</td>
+
       <td class="row">5.2</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E11 (X-P)</td>
+
       <td class="row">E12-1 (X-P)</td>
-
       <td>5.2</td>
+
       <td class="row">5.2</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E12 (X-P)</td>
+
       <td class="row">E16-1 (E-P)</td>
-
       <td>5.2</td>
+
       <td class="row">6.4</td>
   </tr>
   </tr>
-
 
   <tr>
   <tr>
-
       <td>E16 (E-P)</td>
+
       <td class="row">E21-1 (E-P)</td>
-
       <td>6.4</td>
+
       <td class="row">5.8</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E21 (E-P)</td>
+
       <td class="row">E22-2 (E-P)</td>
-
       <td>5.8</td>
+
       <td class="row">6.5</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E22 (E-P)</td>
+
       <td class="row">E23-1 (E-P)</td>
-
       <td>6.5</td>
+
       <td class="row">4.6</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E23 (E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13521">BBa_I13521</a></td>
-
       <td>4.6</td>
+
       <td class="row">6.1</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html><partinfo>BBa_I13521</partinfo><html></td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> in <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61002">BBa_J61002</a> (E-P)</td>
-
      <td>6.1</td>
+
       <td class="row">2.1</td>
-
  </tr>
+
-
 
+
-
  <tr>
+
-
      <td></html><partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo><html></td>
+
-
       <td>2.1</td>
+
   </tr>
   </tr>
</table>
</table>
</center>
</center>
 +
</br>
<p>
<p>
Line 288: Line 261:
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Ligation Name</b></td>
+
       <td class="row"><b>Ligation Name</b></td>
-
       <td><b>Vector</b></td>
+
       <td class="row"><b>Vector</b></td>
-
       <td><b>Vector volume (&mu;l)</b></td>
+
       <td class="row"><b>Vector volume (&mu;l)</b></td>
-
       <td><b>Insert</b></td>
+
       <td class="row"><b>Insert</b></td>
-
       <td><b>Insert volume (&mu;l)</b></td>
+
       <td class="row"><b>Insert volume (&mu;l)</b></td>
-
       <td><b>Buffer (&mu;l)</b></td>
+
       <td class="row"><b>Buffer (&mu;l)</b></td>
-
       <td><b>T4 Ligase (&mu;l)</b></td>
+
       <td class="row"><b>T4 Ligase (&mu;l)</b></td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E24</b></td>
+
       <td class="row"><b>E24</b></td>
-
       <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> (S-P)</td>
-
       <td>1.5</td>
+
       <td class="row">1.5</td>
-
       <td>E9 (X-P)</td>
+
       <td class="row">E9-2 (X-P)</td>
-
       <td>6.5</td>
+
       <td class="row">6.5</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E25</b></td>
+
       <td class="row"><b>E25</b></td>
-
       <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> (S-P)</td>
-
       <td>1.5</td>
+
       <td class="row">1.5</td>
-
       <td>E10 (X-P)</td>
+
       <td class="row">E10-1 (X-P)</td>
-
       <td>6.5</td>
+
       <td class="row">6.5</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E26</b></td>
+
       <td class="row"><b>E26</b></td>
-
       <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> (S-P)</td>
-
       <td>1.5</td>
+
       <td class="row">1.5</td>
-
       <td>E11 (X-P)</td>
+
       <td class="row">E11-1 (X-P)</td>
-
       <td>6.5</td>
+
       <td class="row">6.5</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E27</b></td>
+
       <td class="row"><b>E27</b></td>
-
       <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
+
       <td class="row"></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
-
       <td>1.5</td>
+
       <td class="row">1.5</td>
-
       <td>E12 (X-P)</td>
+
       <td class="row">E12-1 (X-P)</td>
-
       <td>6.5</td>
+
       <td class="row">6.5</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E31</b></td>
+
       <td class="row"><b>E31</b></td>
-
       <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> (E-P)</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>E16 (E-P)</td>
+
       <td class="row">E16-1 (E-P)</td>
-
       <td>5.5</td>
+
       <td class="row">5.5</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E32</b></td>
+
       <td class="row"><b>E32</b></td>
-
       <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
-
       <td>2</td>
+
       <td class="row">2</td>
-
       <td>E21 (E-P)</td>
+
       <td class="row">E21-1 (E-P)</td>
-
       <td>6</td>
+
       <td class="row">6</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E33</b></td>
+
       <td class="row"><b>E33</b></td>
-
       <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
-
       <td>2</td>
+
       <td class="row">2</td>
-
       <td>E22 (E-P)</td>
+
       <td class="row">E22-2 (E-P)</td>
-
       <td>6</td>
+
       <td class="row">6</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E34</b></td>
+
       <td class="row"><b>E34</b></td>
-
       <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
-
       <td>6.5</td>
+
       <td class="row">6.5</td>
-
       <td>E23 (E-P)</td>
+
       <td class="row">E23-1 (E-P)</td>
-
       <td>1.5</td>
+
       <td class="row">1.5</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E35</b></td>
+
       <td class="row"><b>E35</b></td>
-
       <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
-
       <td>2</td>
+
       <td class="row">2</td>
-
       <td></html><partinfo>BBa_I13521</partinfo><html>(E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13521">BBa_I13521</a> (E-P)</td>
-
       <td>6.5</td>
+
       <td class="row">6.5</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td><b>E36</b></td>
+
       <td class="row"><b>E36</b></td>
-
       <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td></html><partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo><html>(E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> in <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61002">BBa_J61002</a> (E-P)</td>
-
       <td>7</td>
+
       <td class="row">7</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
Line 405: Line 378:
<p>
<p>
Ligations were incubated ON at 16°C.
Ligations were incubated ON at 16°C.
 +
<br>
 +
Inoculum of <i>E. coli</i> MGZ1 in 10 ml of LB for competentization.
</p>
</p>
Line 411: Line 386:
</p>
</p>
<a name="July.2C_19th"></a><h2> <span class="mw-headline">July, 19th</span></h2>
<a name="July.2C_19th"></a><h2> <span class="mw-headline">July, 19th</span></h2>
-
<p>
 
<p>
<p>
-
E13 was newly digested for the subsequent gel extraction.
+
According to protocols 200 ml of Buffer 1 and 100 ml of Buffer 2 were prepared for MGZ1 strain competentization.
 +
<br>
 +
E13-1 was newly digested.
</p>
</p>
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Plasmid</b></td>
+
       <td class="row"><b>Plasmid</b></td>
-
       <td><b>Kind</b></td>
+
       <td class="row"><b>Kind</b></td>
-
       <td><b>DNA (&mu;l)</b></td>
+
       <td class="row"><b>DNA (&mu;l)</b></td>
-
       <td><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
+
       <td class="row"><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
-
       <td><b>Enzyme 1 (&mu;l)</b></td>
+
       <td class="row"><b>Enzyme 1 (&mu;l)</b></td>
-
       <td><b>Enzyme 2 (&mu;l)</b></td>
+
       <td class="row"><b>Enzyme 2 (&mu;l)</b></td>
-
       <td><b>Buffer H (&mu;l)</b></td>
+
       <td class="row"><b>Buffer H (&mu;l)</b></td>
-
       <td><b>Final Volume (&mu;l)</b></td>
+
       <td class="row"><b>Final Volume (&mu;l)</b></td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td></html>E13<html></td>
+
       <td class="row"></html>E13<html></td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>6</td>
+
       <td class="row">6</td>
-
       <td>14.5</td>
+
       <td class="row">14.5</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 EcoRI</td>
-
       <td>1 Pstl</td>
+
       <td class="row">1 Pstl</td>
-
       <td>2.5</td>
+
       <td class="row">2.5</td>
-
       <td>25</td>
+
       <td class="row">25</td>
   </tr>
   </tr>
Line 445: Line 421:
<p>
<p>
-
After electrophoresis, E13 insert was succesfully identified and gel extracted (not shown). Meanwhile, sufficient amount of buffer 1 and buffer 2 for MGZ1 competent cell preparation protocol were prepared. E13 DNA was then quantified:
+
After gel electrophoresis, E13-1 insert was succesfully identified.
 +
</p>
 +
 
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_19_07_2011_E13-EPagain.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/UNIPV_19_07_2011_E13-EPagain.png" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Small size gel</div></div></div></div>
 +
 
 +
<p>
 +
E13-1 digested DNA was gel-extracted and quantified:
</p>
</p>
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Plasmid</b></td>
+
       <td class="row"><b>Part</b></td>
-
       <td><b>DNA (ng/&mu;l)</b></td>
+
       <td class="row"><b>DNA (ng/&mu;l)</b></td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E13 (X-P)</td>
+
       <td class="row">E13-1 (E-P)</td>
-
       <td>43</td>
+
       <td class="row">4.3</td>
   </tr>
   </tr>
Line 464: Line 446:
<p>
<p>
-
After E13 digestion (E-P), its ligation was performed in a final volume of 10 &mu;l:
+
E28 ligation was performed in a final volume of 10 &mu;l:
</p>
</p>
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Ligation Name</b></td>
+
       <td class="row"><b>Ligation Name</b></td>
-
       <td><b>Vector</b></td>
+
       <td class="row"><b>Vector</b></td>
-
       <td><b>Vector volume (&mu;l)</b></td>
+
       <td class="row"><b>Vector volume (&mu;l)</b></td>
-
       <td><b>Insert</b></td>
+
       <td class="row"><b>Insert</b></td>
-
       <td><b>Insert volume (&mu;l)</b></td>
+
       <td class="row"><b>Insert volume (&mu;l)</b></td>
-
       <td><b>Buffer (&mu;l)</b></td>
+
       <td class="row"><b>Buffer (&mu;l)</b></td>
-
       <td><b>T4 Ligase (&mu;l)</b></td>
+
       <td class="row"><b>T4 Ligase (&mu;l)</b></td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E28</b></td>
+
       <td class="row"><b>E28</b></td>
-
       <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
-
       <td>6</td>
+
       <td class="row">2</td>
-
       <td>E13 (X-P)</td>
+
       <td class="row">E13 (X-P)</td>
-
       <td>2</td>
+
       <td class="row">6</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
Line 493: Line 475:
<p>
<p>
-
Ligations were incubated ON at 16°C.
+
Ligation was incubated ON at 16°C.
</p>
</p>
<p>
<p>
-
250 ml of M9 were prepared, according to protocols.
+
250 ml of M9 were prepared, according to protocols.<br>Competent MGZ1 cells were prepared according to protocols.
</p>
</p>
<p><a name="indice"/>
<p><a name="indice"/>
</p>
</p>
 +
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<a name="July.2C_20th"></a><h2> <span class="mw-headline">July, 20th</span></h2>
<a name="July.2C_20th"></a><h2> <span class="mw-headline">July, 20th</span></h2>
<p>
<p>
 +
E24, E25, E26 and E27, E36 were transformed in 100 &mu;l of TOP10 competent cells according to protocols. Moreover E28, E31, E32, E33, E34, E35 and <a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0032">BBa_B0032</a> (4 ng of DNA to test transformation efficiency) were transformed in 100 &mu;l of MGZ1 competent cells according to protocols; a negative control was also plated in order to avoid the presence of contaminants. Plates were incubated ON at 37°C.
 +
<br>
 +
In the afternoon 33 LB + Cm 12.5 plates were prepared.
 +
</p>
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="July.2C_21st"></a><h2> <span class="mw-headline">July, 21st</span></h2>
<p>
<p>
-
E24, E25, E26, E27 and were transformed in 100 &mu;l of TOP10 competent cells according to protocols. Moreover E28, E31, E32, E33, E34, E35, E36 and </html><partinfo>B0032</partinfo><html> (transformation efficiency positive control) were transformed in 100 &mu;l of MGZ1 competent cells according to protocols. Plates were incubated ON at 37°C.<br>
+
All plates showed a lot of colonies, except for E28, E31 (2 colonies) and E36; two colonies for each of them were picked, inoculated and screened by PCR. Positive control plate for MGZ1 showed few colonies due to poor transformation efficiency (new comptetent MGZ1 will be prepared!); no colonies grew on negative control plate.
</p>
</p>
<p>
<p>
-
500 ml of LB with chloramphenicol 12.5 were prepared.
+
A 26x mix was prepared in order to perform PCR on E24-1, E24-2, E25-1, E25-2, E26-1, E26-2, E27-1, E27-2, E28-1, E28-2, E31-1, E31-2, E32-1, E32-2, E33-1, E33-2, E34-1, E34-2, E35-1, E35-2, E36-1, E36-2:
 +
</p>
 +
 
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
 +
      <td class="row"><b>Buffer 10x (&mu;l)</b></td>
 +
      <td class="row"><b>MgCl<small><sub>2</sub></small> (&mu;l)</b></td>
 +
      <td class="row"><b>VF2 (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_G00100">BBa_G00100</a>) &mu;l</b></td>
 +
      <td class="row"><b>VR (<a href="http://partsregistry.org/wiki/index.php/Part:BBa_G00101">BBa_G00101</a>) &mu;l</b></td>
 +
      <td class="row"><b>dNTPs (&mu;l)</b></td>
 +
      <td class="row"><b>Taq polymerase (&mu;l)</b></td>
 +
  </tr>
 +
 
 +
    <tr>
 +
      <td class="row">468</td>
 +
      <td class="row">65</td>
 +
      <td class="row">26</td>
 +
      <td class="row">13</td>
 +
      <td class="row">13</td>
 +
      <td class="row">13</td>
 +
      <td class="row">26</td>
 +
  </tr>
 +
 
 +
</table>
 +
</center>
 +
 
 +
<p>
 +
24 &mu;l were aliquoted in each tube, together with 1 &mu;l of each liquid culture. PCR cycles parameters were set as follows:
 +
<ul type="circle">
 +
<li> 94°C 30 seconds (denaturing)
 +
<li> 60°C 1 minute (annealing)
 +
<li> 72°C 1 minute and 30 seconds (elongation)
 +
</ul>
 +
35 cycles were performed.
 +
</p>
 +
 
 +
<p>
 +
Two medium size agarose gels were prepared; in the afternoon gel electrophoresis was carried out:
 +
</p>
 +
 
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_21_07_2011_E24-1_E24-2_E25-1_E25-2_E26-1_E26-2_E27-1_E27-2_E28-1_E28-2_E31-1_Blank.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/0/03/UNIPV_21_07_2011_E24-1_E24-2_E25-1_E25-2_E26-1_E26-2_E27-1_E27-2_E28-1_E28-2_E31-1_Blank.png" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Medium size gel</div></div></div></div>
 +
 
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_21_07_2011_Marker_PCR_E31-2_E32-1_E32-2_E33-1_E33-2_E34-1_E34-2_E35-1_E35-2_E36-1_E36-2_BLANK.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/b/bc/UNIPV_21_07_2011_Marker_PCR_E31-2_E32-1_E32-2_E33-1_E33-2_E34-1_E34-2_E35-1_E35-2_E36-1_E36-2_BLANK.png" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Medium size gel</div></div></div></div>
 +
 
 +
 
 +
<p>
 +
All the band lengths were correct, except E31-2, E36-1 and E36-2 (E36 was plated on the wrong antibiotic!).<br>
 +
Glycerol stocks were prepared for E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2, E34-1 and E35-2.<br>
 +
Sequencing of E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E19-2, E20-2, E21-1, E22-2 and E23-1 were OK.
 +
</p>
 +
 
 +
 
 +
 
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="July.2C_22nd"></a><h2> <span class="mw-headline">July, 22nd</span></h2>
 +
 
 +
<p>
 +
E36 was transformed in 100 μl of TOP10 competent cells according to protocols. The plate was incubated at room temperature.
 +
E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2 and E34-1 plasmid purification was carried out:
 +
</p>
 +
 
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Plasmid</b></td>
 +
      <td class="row"><b>DNA (ng/&mu;l)</b></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E24-2</td>
 +
      <td class="row">76.8</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E25-1</td>
 +
      <td class="row">75.1</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E26-2</td>
 +
      <td class="row">69.4</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E27-2</td>
 +
      <td class="row">81.4</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E28-1</td>
 +
      <td class="row">18.1</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E31-1</td>
 +
      <td class="row">19.4</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E32-2</td>
 +
      <td class="row">21.0</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E33-2</td>
 +
      <td class="row">23.4</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td class="row">E34-1</td>
 +
      <td class="row">25.8</td>
 +
  </tr>
 +
 
 +
</table>
 +
</center>
 +
 
 +
<p>
 +
E17-2, E18-2, E28-1, E31-1, E32-2, E33-2, E34-1, E35-2 purified DNA was sent for sequencing to BMR Genomics.
 +
<br>
 +
Other samples are ready for next week ligations.
</p>
</p>
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<td align="left"><a href="/Team:UNIPV-Pavia/Calendar/July/settimana3" title="Team:UNIPV-Pavia/Calendar/JuLY/settimana3"> Previous week</a></td>
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<td align="right"><a href="/Team:UNIPV-Pavia/Calendar/July/settimana5" title="Team:UNIPV-Pavia/Calendar/July/settimana3"> Next week</a></td>
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<td align="right"><a href="/Team:UNIPV-Pavia/Calendar/July/settimana5" title="Team:UNIPV-Pavia/Calendar/July/settimana5"> Next week</a></td>
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Latest revision as of 14:35, 17 August 2011

UNIPV TEAM 2011

March
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April
M T W T F S S
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May
M T W T F S S
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16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31

June
M T W T F S S
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13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30

July
M T W T F S S
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11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

August
M T W T F S S
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8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31

September
M T W T F S S
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5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30

October
M T W T F S S
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3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31

JULY: WEEK 4

July, 18th

BBa_R0040 in BBa_J61002 plasmid purification and quantification was carried out:

Plasmid DNA (ng/μl)
BBa_R0040 in BBa_J61002 37.3

Digestions of previously purified plasmids were performed for ligations:

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E9-2 Insert 11.5 9 1 Xbal 1 Pstl 2.5 25
E10-1 Insert 10 10.5 1 Xbal 1 Pstl 2.5 25
E11-1 Insert 4 16.5 1 Xbal 1 Pstl 2.5 25
E12-1 Insert 10 10.5 1 Xbal 1 PstI 2.5 25
E13-1 Insert 6 14.5 1 EcoRl 1 Pstl 2.5 25
E16-1 Insert 4 16.5 1 EcoRl 1 Pstl 2.5 25
E21-1 Insert 7.5 13 1 EcoRl 1 Pstl 2.5 25
E22-2 Insert 10 10.5 1 EcoRl 1 Pstl 2.5 25
BBa_I13521 Insert 12.5 8 1 EcoRl 1 Pstl 2.5 25
BBa_R0040 in BBa_J61002 Vector 20.5 0 1 EcoRl 1 Pstl 2.5 25

Reactions were incubated at 37°C for three hours; meanwhile two medium-size agarose gels were prepared according to protocols.
According to protocols, 120 μl of 1kb Plus DNA ladder were prepared.

In the afternoon gel electrophoresis was performed.

Medium size gel
Medium size gel

As shown in figure, all clones were positive (apart from E13-1 run where it is possible to recognize only a band at approximately 2800 bp, corresponding to the linearized plasmid), so we cut and purified the bands of interest.

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
E9-2 (X-P) 5.9
E10-1 (X-P) 4.2
E11-1 (X-P) 5.2
E12-1 (X-P) 5.2
E16-1 (E-P) 6.4
E21-1 (E-P) 5.8
E22-2 (E-P) 6.5
E23-1 (E-P) 4.6
BBa_I13521 6.1
BBa_R0040 in BBa_J61002 (E-P) 2.1

Then ligations were performed in a final volume of 10 μl:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E24 BBa_R0040 (S-P) 1.5 E9-2 (X-P) 6.5 1 1
E25 BBa_R0040 (S-P) 1.5 E10-1 (X-P) 6.5 1 1
E26 BBa_R0040 (S-P) 1.5 E11-1 (X-P) 6.5 1 1
E27 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E12-1 (X-P) 6.5 1 1
E31 BBa_R0040 (E-P) 2.5 E16-1 (E-P) 5.5 1 1
E32 pSB4C5 (E-P) 2 E21-1 (E-P) 6 1 1
E33 pSB4C5 (E-P) 2 E22-2 (E-P) 6 1 1
E34 pSB4C5 (E-P) 6.5 E23-1 (E-P) 1.5 1 1
E35 pSB4C5 (E-P) 2 BBa_I13521 (E-P) 6.5 1 1
E36 pSB4C5 (E-P) 1 BBa_R0040 in BBa_J61002 (E-P) 7 1 1

Ligations were incubated ON at 16°C.
Inoculum of E. coli MGZ1 in 10 ml of LB for competentization.

July, 19th

According to protocols 200 ml of Buffer 1 and 100 ml of Buffer 2 were prepared for MGZ1 strain competentization.
E13-1 was newly digested.

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E13 Insert 6 14.5 1 EcoRI 1 Pstl 2.5 25

After gel electrophoresis, E13-1 insert was succesfully identified.

Small size gel

E13-1 digested DNA was gel-extracted and quantified:

Part DNA (ng/μl)
E13-1 (E-P) 4.3

E28 ligation was performed in a final volume of 10 μl:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E28 pSB4C5 (E-P) 2 E13 (X-P) 6 1 1

Ligation was incubated ON at 16°C.

250 ml of M9 were prepared, according to protocols.
Competent MGZ1 cells were prepared according to protocols.

July, 20th

E24, E25, E26 and E27, E36 were transformed in 100 μl of TOP10 competent cells according to protocols. Moreover E28, E31, E32, E33, E34, E35 and BBa_B0032 (4 ng of DNA to test transformation efficiency) were transformed in 100 μl of MGZ1 competent cells according to protocols; a negative control was also plated in order to avoid the presence of contaminants. Plates were incubated ON at 37°C.
In the afternoon 33 LB + Cm 12.5 plates were prepared.

July, 21st

All plates showed a lot of colonies, except for E28, E31 (2 colonies) and E36; two colonies for each of them were picked, inoculated and screened by PCR. Positive control plate for MGZ1 showed few colonies due to poor transformation efficiency (new comptetent MGZ1 will be prepared!); no colonies grew on negative control plate.

A 26x mix was prepared in order to perform PCR on E24-1, E24-2, E25-1, E25-2, E26-1, E26-2, E27-1, E27-2, E28-1, E28-2, E31-1, E31-2, E32-1, E32-2, E33-1, E33-2, E34-1, E34-2, E35-1, E35-2, E36-1, E36-2:

H2O (μl) Buffer 10x (μl) MgCl2 (μl) VF2 (BBa_G00100) μl VR (BBa_G00101) μl dNTPs (μl) Taq polymerase (μl)
468 65 26 13 13 13 26

24 μl were aliquoted in each tube, together with 1 μl of each liquid culture. PCR cycles parameters were set as follows:

  • 94°C 30 seconds (denaturing)
  • 60°C 1 minute (annealing)
  • 72°C 1 minute and 30 seconds (elongation)
35 cycles were performed.

Two medium size agarose gels were prepared; in the afternoon gel electrophoresis was carried out:

Medium size gel
Medium size gel

All the band lengths were correct, except E31-2, E36-1 and E36-2 (E36 was plated on the wrong antibiotic!).
Glycerol stocks were prepared for E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2, E34-1 and E35-2.
Sequencing of E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E19-2, E20-2, E21-1, E22-2 and E23-1 were OK.

July, 22nd

E36 was transformed in 100 μl of TOP10 competent cells according to protocols. The plate was incubated at room temperature. E24-2, E25-1, E26-2, E27-2, E28-1, E31-1, E32-2, E33-2 and E34-1 plasmid purification was carried out:

Plasmid DNA (ng/μl)
E24-2 76.8
E25-1 75.1
E26-2 69.4
E27-2 81.4
E28-1 18.1
E31-1 19.4
E32-2 21.0
E33-2 23.4
E34-1 25.8

E17-2, E18-2, E28-1, E31-1, E32-2, E33-2, E34-1, E35-2 purified DNA was sent for sequencing to BMR Genomics.
Other samples are ready for next week ligations.