Team:UNIPV-Pavia/Calendar/July/settimana2

From 2011.igem.org

(Difference between revisions)
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       <td><b>Plasmid</b></td>
       <td><b>Plasmid</b></td>
       <td><b>Kind</b></td>
       <td><b>Kind</b></td>
-
       <td><b>Purified Dna (&mu;l)</b></td>
+
       <td><b>Purified DNA (&mu;l)</b></td>
       <td><b>H<small><sub>2</sub></small>O (&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 1 (&mu;l)</b></td>
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<p>
<p>
-
Reaction samples 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 and a medium-size agarose gel were prepared according to protocols.
</p>
</p>
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</center>
</center>
-
As shown, in figure all clones were positive (apart from </html><partinfo>BBa_I13501</partinfo><html> run where we inexplicably didn't see any band), so we cut and purified the bands of interest.
+
<p>
 +
As shown, in figure all clones were positive (apart from </html><partinfo>BBa_I13501</partinfo><html> run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring </html><partinfo>BBa_I13501</partinfo><html> were again inoculated in 5 ml LB + Amp.
 +
</p>
 +
 
 +
<p>
 +
After gel extraction, cut DNA was quantified:
 +
</p>
 +
 
 +
 
 +
 
 +
<center>
 +
<table border="1">
 +
    <tr>
 +
      <td><b>Plasmid</b></td>
 +
      <td><b>Purified DNA ng/&mu;l</b></td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td></html><partinfo>BBa_C0060 (E-S)</partinfo><html></td>
 +
      <td>3.9</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td></html><partinfo>BBa_C0061 (X-P)</partinfo><html></td>
 +
      <td>4.1</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td></html><partinfo>BBa_K081022 (E-P)</partinfo><html></td>
 +
      <td>4.4</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td></html><partinfo>BBa_B0030 (S-P)</partinfo><html></td>
 +
      <td>10.3</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td></html><partinfo>BBa_B0031 (S-P)</partinfo><html></td>
 +
      <td>11.8</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td></html><partinfo>BBa_B0032 (S-P)</partinfo><html></td>
 +
      <td>10.1</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td></html><partinfo>BBa_B0015 (E-X)</partinfo><html></td>
 +
      <td>12</td>
 +
  </tr>
 +
 
 +
 
 +
  <tr>
 +
      <td></html><partinfo>pSB4C5 (E-P)</partinfo><html></td>
 +
      <td>10.7</td>
 +
  </tr>
 +
 
 +
</table>
 +
</center>
 +
 
 +
<p>
 +
Then ligations were performed in a final volume of 10 &mu;l:
 +
</p>
 +
 
 +
<center>
 +
<table border="1">
 +
    <tr>
 +
      <td><b>Ligation Name</b></td>
 +
      <td><b>Vector</b></td>
 +
      <td><b>Vector &mu;l</b></td>
 +
      <td><b>Insert</b></td>
 +
      <td><b>Insert &mu;l</b></td>
 +
      <td><b>Buffer &mu;l</b></td>
 +
      <td><b>T4 Ligase &mu;l</b></td>
 +
  </tr>
 +
 
 +
    <tr>
 +
      <td><b>E1</b></td>
 +
      <td></html><partinfo>BBa_B0015</partinfo><html> (E-X)</td>
 +
      <td>1.5</td>
 +
      <td></html><partinfo>BBa_C0060</partinfo><html> (E-S)</td>
 +
      <td>6.5</td>
 +
      <td><b>1</b></td>
 +
      <td><b>1</b></td>
 +
  </tr>
 +
 
 +
 
 +
    <tr>
 +
      <td><b>E2</b></td>
 +
      <td></html><partinfo>BBa_B0030</partinfo><html> (S-P)</td>
 +
      <td>1.5</td>
 +
      <td></html><partinfo>BBa_C0061</partinfo><html> (X-P)</td>
 +
      <td>6.5</td>
 +
      <td><b>1</b></td>
 +
      <td><b>1</b></td>
 +
  </tr>
 +
 
 +
    <tr>
 +
      <td><b>E3</b></td>
 +
      <td></html><partinfo>BBa_B0031</partinfo><html> (S-P)</td>
 +
      <td>1.5</td>
 +
      <td></html><partinfo>BBa_C0061</partinfo><html> (X-P)</td>
 +
      <td>6.5</td>
 +
      <td><b>1</b></td>
 +
      <td><b>1</b></td>
 +
  </tr>
 +
 
 +
    <tr>
 +
      <td><b>E4</b></td>
 +
      <td></html><partinfo>BBa_B0032</partinfo><html> (S-P)</td>
 +
      <td>1.5</td>
 +
      <td></html><partinfo>BBa_C0061</partinfo><html> (X-P)</td>
 +
      <td>6.5</td>
 +
      <td><b>1</b></td>
 +
      <td><b>1</b></td>
 +
  </tr>
 +
 
 +
    <tr>
 +
      <td><b>E8</b></td>
 +
      <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
 +
      <td>1.5</td>
 +
      <td></html><partinfo>BBa_K081022</partinfo><html> (E-P)</td>
 +
      <td>6.5</td>
 +
      <td><b>1</b></td>
 +
      <td><b>1</b></td>
 +
  </tr>
 +
 
 +
</table>
 +
</center>
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<div align="right"><small><a href="#indice" title="">^top</a></small></div>

Revision as of 16:34, 16 July 2011

UNIPV TEAM 2011

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31

JULY: WEEK 2

July, 4th

Digestions of previously purified plasmids were performed for ligations:

Plasmid Kind Purified DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume(μl)
<partinfo>BBa_C0060</partinfo> Insert 16.5 4 1 EcoRI 1 SpeI 2.5 25
<partinfo>BBa_C0061</partinfo> Insert 13.2 7.3 1 XbaI 1 PstI 2.5 25
<partinfo>BBa_K081022</partinfo> Insert 15.7 4.8 1 EcoRI 1 PstI 2.5 25
<partinfo>BBa_B0030</partinfo> Vector 13.2 7.3 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0031</partinfo> Vector 12.4 8.1 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0032</partinfo> Vector 9.5 11 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0015</partinfo> Vector 7.9 12.6 1 EcoRI 1 XbaI 2.5 25
<partinfo>pSB4C5</partinfo> Vector 3 17.5 1 EcoRI 1 PstI 2.5 25
<partinfo>BBa_I13501</partinfo> Insert 7.2 13.3 1 XbaI 1 PstI 2.5 25

Reactions were incubated at 37°C for three hours while a small-size and a medium-size agarose gel were prepared according to protocols.

In the afternoon gel electrophoresis was performed.

Small size gel
Medium size gel

As shown, in figure all clones were positive (apart from <partinfo>BBa_I13501</partinfo> run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring <partinfo>BBa_I13501</partinfo> were again inoculated in 5 ml LB + Amp.

After gel extraction, cut DNA was quantified:

Plasmid Purified DNA ng/μl
<partinfo>BBa_C0060 (E-S)</partinfo> 3.9
<partinfo>BBa_C0061 (X-P)</partinfo> 4.1
<partinfo>BBa_K081022 (E-P)</partinfo> 4.4
<partinfo>BBa_B0030 (S-P)</partinfo> 10.3
<partinfo>BBa_B0031 (S-P)</partinfo> 11.8
<partinfo>BBa_B0032 (S-P)</partinfo> 10.1
<partinfo>BBa_B0015 (E-X)</partinfo> 12
<partinfo>pSB4C5 (E-P)</partinfo> 10.7

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

Ligation Name Vector Vector μl Insert Insert μl Buffer μl T4 Ligase μl
E1 <partinfo>BBa_B0015</partinfo> (E-X) 1.5 <partinfo>BBa_C0060</partinfo> (E-S) 6.5 1 1
E2 <partinfo>BBa_B0030</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E3 <partinfo>BBa_B0031</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E4 <partinfo>BBa_B0032</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E8 <partinfo>pSB4C5</partinfo> (E-P) 1.5 <partinfo>BBa_K081022</partinfo> (E-P) 6.5 1 1
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