Team:Exeter/lab book/novpol/wk7

From 2012.igem.org

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     <font face="Verdana" color="#57b947" size="2">
        
        
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      <!--Project Division Links-->
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    <!--Project Division Links-->
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       <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
+
      <a href="https://2012.igem.org/Team:Exeter/lab_book/proto"; style="color:#57b947">Protocols</a>
-
        &nbsp;|&nbsp;
+
       &nbsp;|&nbsp;
-
      <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947">Showcasing Polysaccharide Production</a>
+
      <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
-
        &nbsp;|&nbsp;
+
      &nbsp;|&nbsp;
-
      <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947">The 3-Gene Inducible Plasmid</a>
+
      <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947">Showcasing Polysaccharide Production</a>
-
        <p>  
+
      &nbsp;|&nbsp;
-
      <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947">Operon Construction</a>       
+
      <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947">The 3-Gene Inducible Plasmid</a>
-
        &nbsp;|&nbsp;
+
      <p>  
-
      <a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947">Glycobase</a>
+
      <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947">Operon Construction</a>       
-
        </p>
+
      &nbsp;|&nbsp;
-
      <!--End Project Division Links-->
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947">Glycobase</a>
 +
      </p>
 +
    <!--End Project Division Links-->
 +
 
     </font>
     </font>
     </div>
     </div>
   </td>
   </td>
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   </tr>
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   <tr>
   <tr>
     <td rowspan="2" valign="top" align="center" width="170">
     <td rowspan="2" valign="top" align="center" width="170">
      
      
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    <!--Project Division Week Hyperlinks-->
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  <!--Project Division Week Hyperlinks-->
     <div style="text-align:center; width:170">
     <div style="text-align:center; width:170">
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       <font face="DokChampa" color="#1d1d1b" size="3">
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       <font face="Verdana" color="#1d1d1b" size="2">
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#1d1d1b">9th - 13th July</a>
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#1d1d1b">9th - 13th July</a>
         <p>
         <p>
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         -
         -
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk4"; style="color:#1d1d1b">30th July - 3rd August</a>
+
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk5"; style="color:#1d1d1b">6th - 10th August</a>
         <p>
         <p>
         -
         -
         </p>
         </p>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk5"; style="color:#1d1d1b">6th - 10th August</a>
+
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk6"; style="color:#1d1d1b">13th - 17th August</a>
         <p>
         <p>
         -
         -
         </p>
         </p>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk6"; style="color:#1d1d1b">13th - 24th August</a>
+
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk7"; style="color:#1d1d1b">20th - 24th August</a>
         <p>
         <p>
         -
         -
         </p>
         </p>
-
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk7"; style="color:#1d1d1b">27th - 31st August</a>
+
         <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk8"; style="color:#1d1d1b">27th - 31st August</a>
 +
        <p>
 +
        -
 +
        </p>
 +
        <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk9"; style="color:#1d1d1b">3rd - 7th September</a>
 +
        <p>
 +
        -
 +
        </p>
 +
        <a href="https://2012.igem.org/Team:Exeter/Results/showcase"; style="color:#e30614" target="_blank"><font size="3"><b>Results</b></font></a>
 +
</font>
       </font>
       </font>
     </div>
     </div>
     <!--End Project Division Week Hyperlinks-->
     <!--End Project Division Week Hyperlinks-->
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     </td>
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   <td width="680" height="250">
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   <td width="810" height="250">
   <!------------INSERT WEEKLY IMAGE HERE------------>
   <!------------INSERT WEEKLY IMAGE HERE------------>
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     <img src="" alt="" title="" width="680" height="250">
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     <img src="https://static.igem.org/mediawiki/2012/6/6c/Exe2012_aug_20th24th.jpg" alt="" title="" width="810" height="250">
   </td>
   </td>
   </tr>
   </tr>
    
    
   <tr>
   <tr>
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   <td valign="top" width="680">
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   <td valign="top" width="810">
     <div style="text-align:justify">
     <div style="text-align:justify">
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     <font face="Verdana" color="#1d1d1b" size="2">
      
      
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       <font face="DokChampa" color="#57b947" size="4">
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       <font face="Verdana" color="#57b947" size="4">
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       <p><b><u>Showcasing Polysaccharide Production: 27th - 31st August 2012</u></b></p>
+
       <p><b><u>Showcasing Polysaccharide Production: 20th - 24th August 2012</u></b></p>
       </font>
       </font>
     <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------>  
     <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------>  
 +
<p>**<b>Monday 20/08/12</b>**</p><br>
-
<br>
+
<p>Went through all previous constructs and mini-peps to figure out what had worked and what hadn’t. Performed a digest and ran them all on a gel using electrophoresis.</p>
-
<b><p>Thursday 30/08/12</p><br>
+
<p>None of the constructs had worked correctly as they only single banded on the gel.</p><br>
-
 
+
<p>**<b>Tuesday 21/08/12</b>**</p><br>
-
<p>9.30am</p></b>
+
<p>Digestion and ligation of the original HAS, cyclodextran and sacB onto a terminator, into digested tetracycline plasmids using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>3A Assembly</u></a>. The ligated BioBricks were <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>transformed</u></a> onto plates that contained the tetracycline antibiotic. ompA oligomers were <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/5" style="color:#57b947"><u>annealed </u></a>
-
<p>Checked plates. Colonies have formed on all.</p>
+
together in the PCR machine. The annealed ompA was then ligated into a chloramphenicol plasmid and transformed also. </p><br>
-
<p>These were then all placed in the fridge, 4°C.</p>
+
<p>**<b>Wednesday 22/08/12</b>**</p><br>
-
 
+
<p>None of the colonies transformed yesterday had grown overnight. Repeated the digestion and ligation procedure from yesterday again but left them to digest in the incubator for an hour and left them to ligate at room temperature for two hours. Transformed and left overnight again.</p><br>
-
<p><b>4.30pm</b></p>
+
<p>**<b>Thursday 23/08/12</b>**</p><br>
-
<p>Made up liquid broth using plates:
+
<p>The colonies had grown overnight on the plates except for ompA. <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>Transferred</u></a> the constructs to liquid broth and placed in a shaking incubator overnight. The remaining ligation mix of OmpA (7ul) from previously was added to cells and transformed.</p><br>
-
<ul>* pSB1C3 cyclodextrin </ul>
+
<p>**<b>Friday 24/08/12</b>**</p><br>
-
<ul>* pSB1C3 hyaluronan synthase </ul>
+
<p><a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57b947" target="_blank"><u>Mini prep</u></a> of the constructs HAS-terminator, cyclo-terminator and sacB-terminator. Confirmed nucleic acid present using the nanodrop machine. Digested the fragments using the <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>3A Assembly</u></a> protocol and ran on a gel for electrophoresis. Several bands obtained but the ligation had not worked. </p>
-
<ul>* pSB1C3 cyclodextrin + terminator</ul>
+
<p>ompA had successfully grown on the plate so sealed the plate and placed in the fridge. </p><br>
-
<ul>* pSB1C3 hyaluronan synthase  + terminator </ul>
+
-
<ul>* pSB1C3 sacB + terminator </ul>
+
-
 
+
-
<p>4x made per plate – aseptic technique</p>
+
-
<p> Used 10ml broth</p>
+
-
<p> 10μl chloramphenicol</p>
+
-
<p>Scraped off single colony using pipette tip which is then ejected into liquid broth.</p>
+
-
 
+
-
<p><b>5.30pm</b></p>
+
-
<p>Put into horizontal shaker set at 37°C, 220rpm – left overnight.
+
-
<br><br>
+
-
<b><p>Friday 31/08/12</p><br>
+
-
 
+
-
<p>9.30am</p></b>
+
-
<p>Liquid broth transferred to new containers – leaving pipette tip behind.</p>
+
-
<p>These were then centrifuged at 3900rpg for 10 minutes.</p>
+
-
<ul><p><b><i>1.</i></b> supernatant discarded leaving pellet at the base.</ul>
+
-
<ul><b><i>2.</i></b> re-suspended pellet in 250μl re-suspension buffer, used up/down pipette mixing. Moved into eppendorf tubes.</ul>
+
-
<ul><b><i>3.</i></b> added 250μl lysis buffer, mixed each by turning (upside down and back). </ul>
+
-
<ul><b><i>4.</i></b> added 350μl neutralisation buffer. </ul>
+
-
<ul><b><i>5.</i></b> centrifuged at full speed (13k) for 5 minutes. White build up had formed itself in the centre of the vials, centrifuged again for 5 minutes. Slight change, clear fluid became accessible.</ul>
+
-
<ul><b><i>6.</i></b> clear fluid transferred to flow through tubes. </ul>
+
-
<ul><b><i>7.</i></b> centrifuged for 1 minute --> flow through fluid discarded --> 500μl of wash solution added. </ul>
+
-
<ul><b><i>8.</i></b> centrifuged for 1 minute --> flow through fluid discarded --> 500μl of wash solution added again. </ul>
+
-
<ul><b><i>9.</i></b> centrifuged for 1 minute --> flow through fluid discarded --> centrifuged for 1 minute.</ul>
+
-
<ul><b><i>10.</i></b> transferred column to clean eppendorf.</ul>
+
-
<ul><b><i>11.</i></b> added 50μl clean water --> left for 2 minutes --> centrifuged for 2 minutes, kept vial, discarded column.</p></ul>
+
-
<br>
+
-
<b><p>12.30am</p></b>
+
-
<p>Samples were then placed on the NanoDrop machine to get the concentration of DNA.</p>
+
-
<p>Using this and the measurements from the previous digestion, seen below in table 1.</p>  <br>
+
-
 
+
-
 
+
-
<TABLE BORDER="3"  ALIGN="CENTER"  WIDTH="50%"  CELLPADDING="4" CELLSPACING="3">
+
-
 
+
-
<TR>
+
-
      <TH COLSPAN="2"><BR><H3>Table 1. Digest Measurements</H3>
+
-
      </TH>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TH><b>DNA</b></TH>
+
-
      <TD>500ng</TD>
+
-
     
+
-
 
+
-
 
+
-
  </TR>
+
-
<TR ALIGN="CENTER">
+
-
      <TH><b>BUFFER (10x)</b></TH>
+
-
      <TD>2μl</TD>
+
-
 
+
-
 
+
-
 
+
-
  </TR>
+
-
<TR ALIGN="CENTER">
+
-
      <TH><b>BSA (100x)</b></TH>
+
-
      <TD>0.2μl</TD>
+
-
 
+
-
  </TR>
+
-
<TR ALIGN="CENTER">
+
-
      <TH><b>Water</b></TH>
+
-
      <TD>up to 20μl total V</TD>
+
-
 
+
-
 
+
-
  </TR>
+
-
<TR ALIGN="CENTER">
+
-
      <TH><b>ENZYME 1 - ECORI</b></TH>
+
-
      <TD>0.5μL</TD>
+
-
 
+
-
 
+
-
 
+
-
  </TR>
+
-
<TR ALIGN="CENTER">
+
-
    <TH><b>ENZYME 2 - PSTI</b></TH>
+
-
      <TD>0.5μL</TD>
+
-
  </TR>
+
-
</TABLE>
+
-
<br>
+
-
<p>The amount of DNA and water required for all samples could be calculated. </p><br>
+
-
 
+
-
<TABLE BORDER="3"    ALIGN="CENTER" WIDTH="50%"  CELLPADDING="4" CELLSPACING="3">
+
-
 
+
-
  <TR>
+
-
      <TH>Sample</TH>
+
-
      <TH>Conc of DNA </TH>
+
-
      <TH>DNA required</TH>
+
-
      <TH>Water required</TH>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>CYCLODEXTRIN</b></TD>
+
-
      <TD>ng/μl </TD>
+
-
      <TD>μl </TD>
+
-
      <TD>μl </TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>1</b></TD>
+
-
      <TD>40.7</TD>
+
-
      <TD>2.29</TD>
+
-
      <TD>4.51</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>2</b></TD>
+
-
      <TD>55.8</TD>
+
-
      <TD>8.93</TD>
+
-
      <TD>7.90</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>3</b></TD>
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-
      <TD>41.1 </TD>
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-
      <TD> 12.2</TD>
+
-
      <TD> 4.6</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>4</b></TD>
+
-
      <TD>947.5</TD>
+
-
      <TD>0.53</TD>
+
-
      <TD>16.3</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>HYALURONAN SYNTHASE</b></TD>
+
-
      <TD>ng/μl </TD>
+
-
      <TD>μl </TD>
+
-
      <TD>μl </TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>1</b></TD>
+
-
      <TD>194.1</TD>
+
-
      <TD>2.58</TD>
+
-
      <TD>14.22</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>2</b></TD>
+
-
      <TD>106.6</TD>
+
-
      <TD>4.69</TD>
+
-
      <TD>12.11</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>3</b></TD>
+
-
      <TD>40.0 </TD>
+
-
      <TD> 12.5</TD>
+
-
      <TD> 4.3</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>4</b></TD>
+
-
      <TD>94155.4</TD>
+
-
      <TD>3.22</TD>
+
-
      <TD>13.58</TD>
+
-
  </TR>
+
-
 
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>CYCLODEXTRIN +TERMINATOR</b></TD>
+
-
      <TD>ng/μl </TD>
+
-
      <TD>μl </TD>
+
-
      <TD>μl </TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>1</b></TD>
+
-
      <TD>100.4</TD>
+
-
      <TD>5.0</TD>
+
-
      <TD>11.8</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>2</b></TD>
+
-
      <TD>142.8</TD>
+
-
      <TD>3.5</TD>
+
-
      <TD>13.3</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>3</b></TD>
+
-
      <TD>16.3 </TD>
+
-
      <TD> 30.7</TD>
+
-
      <TD> 1.0</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>4</b></TD>
+
-
      <TD>110.8</TD>
+
-
      <TD>4.5</TD>
+
-
      <TD>12.3</TD>
+
-
  </TR>
+
-
 
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>HYALURONAN SYNTHASE + TERMINATOR</b></TD>
+
-
      <TD>ng/μl </TD>
+
-
      <TD>μl </TD>
+
-
      <TD>μl </TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>1</b></TD>
+
-
      <TD>104.6</TD>
+
-
      <TD>4.8</TD>
+
-
      <TD>12</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>2</b></TD>
+
-
      <TD>211.7</TD>
+
-
      <TD>2.36</TD>
+
-
      <TD>14.4</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>3</b></TD>
+
-
      <TD>55.5</TD>
+
-
      <TD> 9.0</TD>
+
-
      <TD> 7.8</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>4</b></TD>
+
-
      <TD>70.4</TD>
+
-
      <TD>7.1</TD>
+
-
      <TD>9.7</TD>
+
-
  </TR>
+
-
 
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>SacB + TERMINATOR</b></TD>
+
-
      <TD>ng/μl </TD>
+
-
      <TD>μl </TD>
+
-
      <TD>μl </TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>1</b></TD>
+
-
      <TD>119.3</TD>
+
-
      <TD>4.2</TD>
+
-
      <TD>12.6</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>2</b></TD>
+
-
      <TD>22.5</TD>
+
-
      <TD>22.0</TD>
+
-
      <TD>1.0</TD>
+
-
  </TR>
+
-
 
+
-
<TR ALIGN="CENTER">
+
-
      <TD><b>3</b></TD>
+
-
      <TD>84.0 </TD>
+
-
      <TD> 6.0</TD>
+
-
      <TD> 10.8</TD>
+
-
  </TR>
+
-
 
+
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<TR ALIGN="CENTER">
+
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      <TD><b>4</b></TD>
+
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      <TD>67.9</TD>
+
-
      <TD>7.4</TD>
+
-
      <TD>9.4</TD>
+
-
  </TR>
+
-
 
+
-
 
+
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</TABLE>
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    <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> &nbsp;&nbsp;|&nbsp;&nbsp;
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Latest revision as of 23:58, 26 September 2012

ExiGEM2012 Lab Book NovPol wk7

Showcasing Polysaccharide Production: 20th - 24th August 2012

**Monday 20/08/12**


Went through all previous constructs and mini-peps to figure out what had worked and what hadn’t. Performed a digest and ran them all on a gel using electrophoresis.

None of the constructs had worked correctly as they only single banded on the gel.


**Tuesday 21/08/12**


Digestion and ligation of the original HAS, cyclodextran and sacB onto a terminator, into digested tetracycline plasmids using 3A Assembly. The ligated BioBricks were transformed onto plates that contained the tetracycline antibiotic. ompA oligomers were annealed together in the PCR machine. The annealed ompA was then ligated into a chloramphenicol plasmid and transformed also.


**Wednesday 22/08/12**


None of the colonies transformed yesterday had grown overnight. Repeated the digestion and ligation procedure from yesterday again but left them to digest in the incubator for an hour and left them to ligate at room temperature for two hours. Transformed and left overnight again.


**Thursday 23/08/12**


The colonies had grown overnight on the plates except for ompA. Transferred the constructs to liquid broth and placed in a shaking incubator overnight. The remaining ligation mix of OmpA (7ul) from previously was added to cells and transformed.


**Friday 24/08/12**


Mini prep of the constructs HAS-terminator, cyclo-terminator and sacB-terminator. Confirmed nucleic acid present using the nanodrop machine. Digested the fragments using the 3A Assembly protocol and ran on a gel for electrophoresis. Several bands obtained but the ligation had not worked.

ompA had successfully grown on the plate so sealed the plate and placed in the fridge.


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