Team:UNIPV-Pavia/Calendar/July/settimana5

From 2011.igem.org

(Difference between revisions)
 
(22 intermediate revisions not shown)
Line 15: Line 15:
<p>
<p>
-
Red colonies were observed from E36 plates, suggesting that the transformation was successful. A colony was picked form the plate.
+
Red colonies were observed from E36 plate, suggesting that the transformation was successful. A colony was picked form the plate.
</p>
</p>
Line 24: Line 24:
<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>E24-2</td>
+
       <td class="row">E24-2</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>13</td>
+
       <td class="row">13</td>
-
       <td>7.5</td>
+
       <td class="row">7.5</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 XbaI</td>
-
       <td>1 Pstl</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>E25-1</td>
+
       <td class="row">E25-1</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>13.5</td>
+
       <td class="row">13.5</td>
-
       <td>7</td>
+
       <td class="row">7</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 XbaI</td>
-
       <td>1 Pstl</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>E26-2</td>
+
       <td class="row">E26-2</td>
-
       <td>Insert</td>
+
       <td class="row">Insert</td>
-
       <td>14.5</td>
+
       <td class="row">14.5</td>
-
       <td>6</td>
+
       <td class="row">6</td>
-
       <td>1 Xbal</td>
+
       <td class="row">1 XbaI</td>
-
       <td>1 Pstl</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>E27-1</td>
+
       <td class="row">E27-2</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 Xbal</td>
+
       <td class="row">1 XbaI</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>
Line 87: Line 87:
<p>
<p>
-
Reactions were incubated at 37°C for three hours while a small-size and a medium-size agarose gel were prepared according to protocols.
+
Reactions were incubated at 37°C for three hours while a small size agarose gel was prepared according to protocols.
 +
<br>
 +
In the afternoon gel electrophoresis was performed:
</p>
</p>
-
<p>
 
-
In the afternoon gel electrophoresis was performed.
 
-
</p>
 
-
<p>
+
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_25_07_2011_E24_E-P_E25_E-P_E26_E-P_E27-E-P.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/66/UNIPV_25_07_2011_E24_E-P_E25_E-P_E26_E-P_E27-E-P.png" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Small size gel</div></div></div></div>
-
Immagine della corsa su gel
+
 
-
</p>
+
<p>
<p>
Line 105: Line 103:
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Part</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>E24 (E-P)</td>
+
       <td class="row">E24-2 (E-P)</td>
-
       <td>-</td>
+
       <td class="row">7.4</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E25 (E-P)</td>
+
       <td class="row">E25-1 (E-P)</td>
-
       <td>-</td>
+
       <td class="row">8.4</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E26 (E-P)</td>
+
       <td class="row">E26-2 (E-P)</td>
-
       <td>-</td>
+
       <td class="row">3.2</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E27 (E-P)</td>
+
       <td class="row">E27-2 (E-P)</td>
-
       <td>-</td>
+
       <td class="row">6.3</td>
   </tr>
   </tr>
Line 138: Line 136:
<p>
<p>
-
Previously purified plasmids carrying either E17, or E18, or E19 or E20 part were inoculated in order to amplify them for the subsequent transformation in MGZ1 cells (E' GIUSTO?):
+
Ligations were performed:
</p>
</p>
-
</br>
 
-
<p><a name="indice"/>
+
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Ligation Name</b></td>
 +
      <td class="row"><b>Vector</b></td>
 +
      <td class="row"><b>Vector volume (&mu;l)</b></td>
 +
      <td class="row"><b>Insert</b></td>
 +
      <td class="row"><b>Insert volume (&mu;l)</b></td>
 +
      <td class="row"><b>Buffer (&mu;l)</b></td>
 +
      <td class="row"><b>T4 Ligase (&mu;l)</b></td>
 +
  </tr>
 +
 
 +
    <tr>
 +
      <td class="row"><b>E37</b></td>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
 +
      <td class="row">2</td>
 +
      <td class="row">E24-2 (E-P)</td>
 +
      <td class="row">6</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
 
 +
 
 +
    <tr>
 +
      <td class="row"><b>E38</b></td>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
 +
      <td class="row">2.5</td>
 +
      <td class="row">E25-1 (E-P)</td>
 +
      <td class="row">5.5</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
 
 +
 
 +
    <tr>
 +
      <td class="row"><b>E39</b></td>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
 +
      <td class="row">1</td>
 +
      <td class="row">E26-2 (E-P)</td>
 +
      <td class="row">7</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
 
 +
 
 +
    <tr>
 +
      <td class="row"><b>E40</b></td>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (E-P)</td>
 +
      <td class="row">2</td>
 +
      <td class="row">E27-2 (E-P)</td>
 +
      <td class="row">6</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 
 +
<p>
 +
Inoculum of E17-2, E18-2, E19-2 or E20-2 to amplify DNA and transfer it into MGZ1; refill for E36 liquid culture.<br>
 +
Glycerol stock for E36 was prepared<br>
 +
250 ml of LB + Cm 12.5 were prepared.
</p>
</p>
 +
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<a name="July.2C_26th"></a><h2> <span class="mw-headline">July, 26th</span></h2>
<a name="July.2C_26th"></a><h2> <span class="mw-headline">July, 26th</span></h2>
<p>
<p>
<p>
<p>
-
Plasmids containing either E17, or E18, or E19, or E20 or E36 part were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:
+
Plasmids containing E17-2, E18-2, E19-2, E20-2 and E36 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:
</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>DNA (ng/&mu;l)</b></td>
+
       <td class="row"><b>DNA (ng/&mu;l)</b></td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E17</td>
+
       <td class="row">E17-2</td>
-
       <td>20.3</td>
+
       <td class="row">20.3</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E18</td>
+
       <td class="row">E18-2</td>
-
       <td>18.6</td>
+
       <td class="row">18.6</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E19</td>
+
       <td class="row">E19-2</td>
-
       <td>17.3</td>
+
       <td class="row">17.3</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E20</td>
+
       <td class="row">E20-2</td>
-
       <td>13.2</td>
+
       <td class="row">13.2</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
       <td>E36</td>
+
       <td class="row">E36</td>
-
       <td>17.8</td>
+
       <td class="row">17.8</td>
   </tr>
   </tr>
</table>
</table>
</center>
</center>
 +
<p>
<p>
-
Digestion of E36 part carrying plasmid was performed for ligation:
+
Digestion of E36 was performed for ligations:
</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>E36</td>
+
       <td class="row">E36</td>
-
       <td>Insert</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 Xbal</td>
+
       <td class="row">1 SpeI</td>
-
       <td>1 Pstl</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>
Line 220: Line 281:
<p>
<p>
-
Digestions of previously purified plasmids were performed for ligations:
+
The sample was incubated at 37°C for three hours while a small-size  agarose gel was prepared; in the afteroon gel electrophoresis was carried out:
 +
</p>
 +
 
 +
 
 +
 
 +
<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_26_07_2011_E36_S-P.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d4/UNIPV_26_07_2011_E36_S-P.png" class="thumbimage" height="80%" width="80%"></a><div class="thumbcaption">Small size gel</div></div></div></div>
 +
 
 +
 
 +
<p>
 +
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>E36</td>
+
       <td class="row">E36 (S-P)</td>
-
       <td>Insert</td>
+
       <td class="row">3.5</td>
-
      <td>13</td>
+
-
      <td>7.5</td>
+
-
      <td>1 Xbal</td>
+
-
      <td>1 Pstl</td>
+
-
      <td>2.5</td>
+
-
      <td>25</td>
+
   </tr>
   </tr>
-
 
</table>
</table>
Line 252: Line 309:
<p>
<p>
-
Reactions were incubated at 37°C for three hours while a small-size  agarose gel was prepared according to protocols.
+
Then ligations were performed in a final volume of 10 &mu;l:
</p>
</p>
 +
<center>
 +
<table class="data">
 +
    <tr>
 +
      <td class="row"><b>Ligation Name</b></td>
 +
      <td class="row"><b>Vector</b></td>
 +
      <td class="row"><b>Vector volume (&mu;l)</b></td>
 +
      <td class="row"><b>Insert</b></td>
 +
      <td class="row"><b>Insert volume (&mu;l)</b></td>
 +
      <td class="row"><b>Buffer (&mu;l)</b></td>
 +
      <td class="row"><b>T4 Ligase (&mu;l)</b></td>
 +
  </tr>
 +
 +
    <tr>
 +
      <td class="row"><b>E41</b></td>
 +
      <td class="row">E36 (S-P)</td>
 +
      <td class="row">4</td>
 +
      <td class="row">E3-1 (X-P)</td>
 +
      <td class="row">4</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
 +
 +
    <tr>
 +
      <td class="row"><b>E42</b></td>
 +
      <td class="row">E36 (S-P)</td>
 +
      <td class="row">3.5</td>
 +
      <td class="row">E4-2 (X-P)</td>
 +
      <td class="row">4.5</td>
 +
      <td class="row">1</td>
 +
      <td class="row">1</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
<p>
<p>
-
In the afternoon gel electrophoresis was performed.
+
Ligations were incubated at 16°C ON.
</p>
</p>
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="July.2C_27th"></a><h2> <span class="mw-headline">July, 27th</span></h2>
<p>
<p>
-
Immagine della corsa su gel
+
 
 +
<p>
 +
E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 &mu;l of MGZ1 competent cells according to protocols. Moreover 4ng of purified <a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0032">BBa_B0032</a> DNA were transformed in MGZ1 in order to test the transformation efficiency. Plates were incubated ON at 37°C.<br>
 +
BMR Genomics sent results of parts sequencing; E17-2, E18-2, E31-1, E32-2, E33-2, E34-1, E35-2 were OK while E28-1 sequence was wrong.
</p>
</p>
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="July.2C_28th"></a><h2> <span class="mw-headline">July, 28th</span></h2>
<p>
<p>
-
After gel extraction, digested DNA was quantified:
+
Colonies grew in every plate. In fact E17-2, E18-2, E19-2 and E20-2, E37, E38, E39 and E40 showed grown colonies. We picked two colonies for each plate and inoculate them in 5 ml of LB + Cm 12.5. As regards to E41 and E42 parts, we resolved to let them still grow; in the late morning we picked and inoculate two colonies for both plates.<br>
 +
Plate with <a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0032">BBa_B0032</a> showed 9 colonies; the corresponding efficiency results in 2250 colonies/&mu;g of DNA (very low, we need to prepare new competent MGZ1).
</p>
</p>
 +
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="July.2C_29th"></a><h2> <span class="mw-headline">July, 29th</span></h2>
 +
 +
<p>
 +
Glycerol stocks were prepared for E17-2, E18-2, E19-2, E20-2 in MGZ1.
 +
<br>
 +
Plasmid purification was carried out on over-night grown cultures (E41-1 did not grow!):
 +
</p>
 +
<center>
<center>
-
<table border="1">
+
<table class="data">
     <tr>
     <tr>
-
       <td><b>Part</b></td>
+
       <td class="row"><b>Plasmid</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>E36 (S-P)</td>
+
       <td class="row">E37-1</td>
-
       <td>-</td>
+
       <td class="row">16.6</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E37-2</td>
 +
      <td class="row">16.9</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E38-1</td>
 +
      <td class="row">28.4</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E38-2</td>
 +
      <td class="row">15.0</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E39-1</td>
 +
      <td class="row">15.0</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E39-2</td>
 +
      <td class="row">14.5</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E40-1</td>
 +
      <td class="row">17.1</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E40-2</td>
 +
      <td class="row">38.9</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E41-2</td>
 +
      <td class="row">13.7</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E42-1</td>
 +
      <td class="row">22.7</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td class="row">E42-2</td>
 +
      <td class="row">28</td>
   </tr>
   </tr>
Line 283: Line 454:
<p>
<p>
-
Then ligations were performed in a final volume of 10 &mu;l:
+
A 12x mix was prepared for screening digestions:
</p>
</p>
 +
<center>
<center>
-
<table border="1">
+
<table class="data">
 +
  <tr>
 +
      <td class="row"><b>H<small><sub>2</sub></small>O (μl)</b></td>
 +
      <td class="row"><b>Enzyme 1 (μl)</b></td>
 +
      <td class="row"><b>Enzyme 2 (μl)</b></td>
 +
      <td class="row"><b>Buffer H (μl)</b></td>
 +
  </tr>
 +
 
 +
<tr>
 +
      <td class="row">126</td>
 +
      <td class="row">12 EcoRI</td>
 +
      <td class="row">12 PstI</td>
 +
      <td class="row">30</td>
 +
  </tr>
 +
</table>
 +
</center>
 +
 
 +
<p>
 +
For each digestion 10 &mu;l of purified DNA were used. Meanwhile a medium size gel was prepared.<br>
 +
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_29_07_2011_E37-1_E37-2_E38-1_E38-2_E39-1_E39-2_E40-1_E40-2_E42-2_E42-1_E41-2.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/a/a7/UNIPV_29_07_2011_E37-1_E37-2_E38-1_E38-2_E39-1_E39-2_E40-1_E40-2_E42-2_E42-1_E41-2.png" class="thumbimage" height="80%" width="80%"></a>  <div class="thumbcaption">Medium size gel</div></div></div></div>
 +
 
 +
<p>
 +
All clones were positive except for E42-2 and E41-2.
 +
<br>
 +
Glycerol stocks were prepared for E37-2, E38-1, E39-1, E40-2 and E42-1.<br>
 +
Ligations of consitutive promoter <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a> with different RBS in <a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> were performed:
 +
</p>
 +
 
 +
<center>
 +
<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>E41</b></td>
+
       <td class="row"><b><a href="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>-31</b></td>
-
       <td>E36 (S-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a> in <a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (S-P)</td>
-
       <td>4</td>
+
       <td class="row">4</td>
-
       <td>E3 (X-P)</td>
+
       <td class="row">E6 (X-P)</td>
-
       <td>4.1</td>
+
       <td class="row">4</td>
-
       <td>1</td>
+
      <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
 +
  </tr>
 +
 
 +
 
 +
    <tr>
 +
      <td class="row"><b><a href="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>-E5</b></td>
 +
      <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a> in <a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (S-P)</td>
 +
      <td class="row">4</td>
 +
      <td class="row">E5 (X-P)</td>
 +
      <td class="row">4</td>
 +
      <td class="row">1</td>
 +
       <td class="row">1</td>
   </tr>
   </tr>
     <tr>
     <tr>
-
       <td><b>E42</b></td>
+
       <td class="row"><b><a href="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>-E7</b></td>
-
       <td>E36 (S-P)</td>
+
       <td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a> in <a href="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> (S-P)</td>
-
       <td>3.5</td>
+
       <td class="row">4</td>
-
       <td>E4 (X-P)</td>
+
       <td class="row">E7 (X-P)</td>
-
       <td>4.5</td>
+
       <td class="row">4</td>
-
       <td>1</td>
+
       <td class="row">1</td>
-
       <td>1</td>
+
       <td class="row">1</td>
   </tr>
   </tr>
Line 322: Line 538:
</center>
</center>
-
</br>
 
-
 
-
<p><a name="indice"/>
 
-
</p>
 
-
<a name="July.2C_27th"></a><h2> <span class="mw-headline">July, 27th</span></h2>
 
-
<p>
 
 +
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="July.2C_30th"></a><h2> <span class="mw-headline">July, 30th</span></h2>
<p>
<p>
-
E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 μl of MGZ1 competent cells according to protocols. Moreover a plasmid containing <a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13501">BBa_B0032</a> was transformed in order to test the transformation efficiency. Plates were incubated ON at 37°C.
+
E28 and E41 were again transformed in 200 &mu;l of MGZ1 competent cells.
 +
<br>
 +
J101-E5, J101-31 and J101-E7 ligations were transformed in 100 &mu;l of TOP10 competent cells.
</p>
</p>
-
</br>
 
-
<p><a name="indice"/>
+
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 +
<a name="July.2C_31st"></a><h2> <span class="mw-headline">July, 31st</span></h2>
 +
<p>
 +
All plates showed colonies. One colony was picked from each of J101-E5, J101-31 and J101-E7 plates, inoculated in LB + Cm 12.5 and grown.
</p>
</p>
-
</br>
+
<table border="0" width="100%" class="menu">
 +
<tr>
 +
<td align="left"><a href="/Team:UNIPV-Pavia/Calendar/July/settimana4" title="Team:UNIPV-Pavia/Calendar/JuLY/settimana4"> Previous week</a></td>
 +
<td align="right"><a href="/Team:UNIPV-Pavia/Calendar/August/week1" title="Team:UNIPV-Pavia/Calendar/August/week1"> Next week</a></td>
 +
</tr>
 +
</table>
-
<p><a name="indice"/>
 
-
</p>
 
-
<a name="July.2C_28th"></a><h2> <span class="mw-headline">July, 28th</span></h2>
 
-
<p>
 
-
<p>
 
-
Colonies grew in every plate. Infact both 17-2, E18-2, E19-2 and E20-2 parts, whose DNA was recovered after MiniPrep kit extraction, both E37, E38, E39 and E40 parts showed grown colonies. We picked two colonies from all parts. As regards to E41 and E42 parts, we resolved to let them still grow.<br>
 
-
Plate with <a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13501">BBa_B0032</a> showed 9 colonies. The corresponding efficiency results 9/4 [1/ng] = 2250 [1/&mu;l] 
 
-
</p>
 
</html>
</html>
-
{{end}}
+
{{endcalendar}}

Latest revision as of 14:47, 17 August 2011

UNIPV TEAM 2011

March
M T W T F S S
  1 2 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    

April
M T W T F S S
        1 2 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

May
M T W T F S S
            1
2 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

June
M T W T F S S
    1 2 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

July
M T W T F S S
        1 2 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

August
M T W T F S S
1 2 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

September
M T W T F S S
      1 2 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

October
M T W T F S S
          1 2
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 5

July, 25th

Red colonies were observed from E36 plate, suggesting that the transformation was successful. A colony was picked form the plate.

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)
E24-2 Insert 13 7.5 1 XbaI 1 PstI 2.5 25
E25-1 Insert 13.5 7 1 XbaI 1 PstI 2.5 25
E26-2 Insert 14.5 6 1 XbaI 1 PstI 2.5 25
E27-2 Insert 12.5 8 1 XbaI 1 PstI 2.5 25

Reactions were incubated at 37°C for three hours while a small size agarose gel was prepared according to protocols.
In the afternoon gel electrophoresis was performed:

Small size gel

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
E24-2 (E-P) 7.4
E25-1 (E-P) 8.4
E26-2 (E-P) 3.2
E27-2 (E-P) 6.3

Ligations were performed:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E37 pSB4C5 (E-P) 2 E24-2 (E-P) 6 1 1
E38 pSB4C5 (E-P) 2.5 E25-1 (E-P) 5.5 1 1
E39 pSB4C5 (E-P) 1 E26-2 (E-P) 7 1 1
E40 pSB4C5 (E-P) 2 E27-2 (E-P) 6 1 1

Inoculum of E17-2, E18-2, E19-2 or E20-2 to amplify DNA and transfer it into MGZ1; refill for E36 liquid culture.
Glycerol stock for E36 was prepared
250 ml of LB + Cm 12.5 were prepared.

July, 26th

Plasmids containing E17-2, E18-2, E19-2, E20-2 and E36 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:

Plasmid DNA (ng/μl)
E17-2 20.3
E18-2 18.6
E19-2 17.3
E20-2 13.2
E36 17.8

Digestion of E36 was performed for ligations:

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E36 Vector 20.5 0 1 SpeI 1 PstI 2.5 25

The sample was incubated at 37°C for three hours while a small-size agarose gel was prepared; in the afteroon gel electrophoresis was carried out:

Small size gel

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
E36 (S-P) 3.5

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)
E41 E36 (S-P) 4 E3-1 (X-P) 4 1 1
E42 E36 (S-P) 3.5 E4-2 (X-P) 4.5 1 1

Ligations were incubated at 16°C ON.

July, 27th

E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 μl of MGZ1 competent cells according to protocols. Moreover 4ng of purified BBa_B0032 DNA were transformed in MGZ1 in order to test the transformation efficiency. Plates were incubated ON at 37°C.
BMR Genomics sent results of parts sequencing; E17-2, E18-2, E31-1, E32-2, E33-2, E34-1, E35-2 were OK while E28-1 sequence was wrong.

July, 28th

Colonies grew in every plate. In fact E17-2, E18-2, E19-2 and E20-2, E37, E38, E39 and E40 showed grown colonies. We picked two colonies for each plate and inoculate them in 5 ml of LB + Cm 12.5. As regards to E41 and E42 parts, we resolved to let them still grow; in the late morning we picked and inoculate two colonies for both plates.
Plate with BBa_B0032 showed 9 colonies; the corresponding efficiency results in 2250 colonies/μg of DNA (very low, we need to prepare new competent MGZ1).

July, 29th

Glycerol stocks were prepared for E17-2, E18-2, E19-2, E20-2 in MGZ1.
Plasmid purification was carried out on over-night grown cultures (E41-1 did not grow!):

Plasmid DNA (ng/μl)
E37-1 16.6
E37-2 16.9
E38-1 28.4
E38-2 15.0
E39-1 15.0
E39-2 14.5
E40-1 17.1
E40-2 38.9
E41-2 13.7
E42-1 22.7
E42-2 28

A 12x mix was prepared for screening digestions:

H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl)
126 12 EcoRI 12 PstI 30

For each digestion 10 μl of purified DNA were used. Meanwhile a medium size gel was prepared.
In the afternoon gel electrophoresis was carried out:

Medium size gel

All clones were positive except for E42-2 and E41-2.
Glycerol stocks were prepared for E37-2, E38-1, E39-1, E40-2 and E42-1.
Ligations of consitutive promoter BBa_J23101 with different RBS in pSB4C5 were performed:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
BBa_J23101-31 BBa_J23101 in pSB4C5 (S-P) 4 E6 (X-P) 4 1 1
BBa_J23101-E5 BBa_J23101 in pSB4C5 (S-P) 4 E5 (X-P) 4 1 1
BBa_J23101-E7 BBa_J23101 in pSB4C5 (S-P) 4 E7 (X-P) 4 1 1

July, 30th

E28 and E41 were again transformed in 200 μl of MGZ1 competent cells.
J101-E5, J101-31 and J101-E7 ligations were transformed in 100 μl of TOP10 competent cells.

July, 31st

All plates showed colonies. One colony was picked from each of J101-E5, J101-31 and J101-E7 plates, inoculated in LB + Cm 12.5 and grown.