Team:UC Davis/Notebook/Protocols

From 2012.igem.org

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 +
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 +
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Line 287: Line 801:
font-weight:bold;
font-weight:bold;
font-size: 40px;
font-size: 40px;
-
color: #004b85;;
+
color: #016D8B;;
left:10px;
left:10px;
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Line 323: Line 837:
 +
#firstpane {
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Line 331: Line 895:
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Line 346: Line 1,006:
    <ul>
    <ul>
  <li style='color:#014457; cursor:default'><a>teams</a></li>
  <li style='color:#014457; cursor:default'><a>teams</a></li>
-
                    <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis">Page</a></li>
+
                    <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols">Page</a></li>
-
                 <li class='new'><a href="https://2012.igem.org/wiki/index.php?title=Talk:Team:UC_Davis&amp;action=edit&amp;redlink=1">Discussion</a></li>
+
                 <li class='new'><a href="https://2012.igem.org/wiki/index.php?title=Talk:Team:UC_Davis/Notebook/Protocols&amp;action=edit&amp;redlink=1">Discussion</a></li>
               <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&action=edit">Edit</a></li>
               <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&action=edit">Edit</a></li>
-
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis&amp;action=history">History</a></li>
+
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&amp;action=history">History</a></li>
-
                 <li><a href="https://2012.igem.org/Special:MovePage/Team:UC_Davis">Move</a></li>
+
                 <li><a href="https://2012.igem.org/Special:MovePage/Team:UC_Davis/Notebook/Protocols">Move</a></li>
-
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis&amp;action=watch">Watch</a></li>
+
                           <li><a href="https://2012.igem.org/wiki/index.php?title=Team:UC_Davis/Notebook/Protocols&amp;action=watch">Watch</a></li>
  <li><a href="https://igem.org/Login">Log in</a></li>
  <li><a href="https://igem.org/Login">Log in</a></li>
Line 359: Line 1,019:
   <div id="newnavi">
   <div id="newnavi">
     <ul class="newmenu">
     <ul class="newmenu">
-
        <li ><a href="https://2012.igem.org/" title="Back to iGEM">iGEM</a></li>
+
    <li ><a target="new" href="https://2012.igem.org/" title="Back to iGEM">iGEM</a>
 +
          <ul>
 +
          <li><a target="new" href="https://2012.igem.org/">Main iGEM</a></li>
 +
          <li><a href="https://2012.igem.org/Team:UC_Davis/Criteria">Criteria</a></li>
 +
          <li><a href="https://2012.igem.org/Team:UC_Davis/Human_Practices">Human Practices</a></li>
 +
          </ul>
 +
        </li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Attributions" title="Attributions">Attributions</a></li>
-
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
+
         <li ><a title="https://2012.igem.org/Team:UC_Davis/Data" title="Data">Data</a>
           <ul>
           <ul>
-
             <li ><a href="./Data.htm ">Data 1</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Cutinase_Activity" title="Data">Cutinase Activity</a></li>
-
             <li ><a href="./Data.htm ">Data 2</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Ethylene_Glycol"
-
            <li ><a href="./Data.htm ">Data 3</a></li>
+
title="Data">Ethylene Glycol</a></li>
-
        </ul>
+
<li ><a href="https://2012.igem.org/Team:UC_Davis/Data/Modeling"
 +
title="Data">Modeling</a></li>
 +
 
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Parts">Parts</a></li>
 +
          </ul>
         </li>
         </li>
         <li class="selected"><a href="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
         <li class="selected"><a href="https://2012.igem.org/Team:UC_Davis/Notebook" title="Notebook">Notebook</a>
           <ul>
           <ul>
-
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols">Protocols</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook">Notebook</a></li>
-
             <li ><a href="./Notebook.htm ">Week 1</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Protocols ">Protocols</a></li>
-
             <li ><a href="./Notebook.htm ">Week 2</a></li>
+
             <li ><a href="https://2012.igem.org/Team:UC_Davis/Notebook/Gallery">Gallery</a></li>
-
            <li ><a href="./Notebook.htm ">Week 3</a></li>
+
           </ul>
           </ul>
         </li>
         </li>
Line 381: Line 1,050:
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Safety" title="Safety">Safety</a></li>
-
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a></li>
+
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Project" title="Project">Project</a>
 +
          <ul>
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project">Project Overview</a></li>
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Catalyst">Module Engineering</a></li>
 +
            <li ><a href="https://2012.igem.org/Team:UC_Davis/Project/Protein_Engineering">Protein Engineering</a></li>
 +
            <li ><a title="https://2012.igem.org/Team:UC_Davis/Project/Strain">Chassis Engineering </a>
 +
  <ul>
 +
                <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Strain">Background</a></li>
 +
        <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Directed_Evolution">Directed Evolution</a></li>
 +
                <li><a href="https://2012.igem.org/Team:UC_Davis/Project/Our_Strain">Rational Engineering </a></li>
 +
</ul>
 +
</li>
 +
          </ul>
 +
        </li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Team" title="Team">Team</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis/Team" title="Team">Team</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis" title="Home">Home</a></li>
         <li ><a href="https://2012.igem.org/Team:UC_Davis" title="Home">Home</a></li>
     </ul>
     </ul>
   </div>
   </div>
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<img src="https://static.igem.org/mediawiki/2012/7/71/UCD_protocols_banner.jpg" width="850" height="228">
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 +
<!-- twitter starts here -->
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<div id="tweets">
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<center>
 +
<h2>UCDavis iGEM Tweets</h2>
 +
</center>
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<script charset="utf-8" src="http://widgets.twimg.com/j/2/widget.js"></script>
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<script>
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<img src="http://img.photobucket.com/albums/v26/bluemelon/protocols_banner-1.jpg" width="850" height="214">
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+
<div id="sponsorbox" class="sponsorfloat">
-
  <div class="floatbox3">
+
<center>
-
<h1> Protocols </h1>
+
<h2>Our Sponsors</h2>
-
<article>
+
<a href="http://www.novozymes.com/en/Pages/default.aspx" target="_blank"><img src="https://static.igem.org/mediawiki/2011/2/21/UCD_Novozymes-logo.jpg" width="200"></a>
-
These are our protocols.
+
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-
</article>
+
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+
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<br>
+
<center>
-
  <div class="floatbox3">
+
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-
<article>
+
</center>
-
<hp> Registry Part Distribution Rehydration</hp>
+
-
<br>•Add 20ul sterile H2O to desired well in distribution plate.
+
-
<br>•Incubate at room temperature for ~10 minutes.
+
-
<br>•Transfer resuspension to microcentrifuge tube.
+
-
</article></div>
+
-
<br><div class="floatbox3">
+
<center>
-
<article>
+
<a href="http://biosci.ucdavis.edu/index_js.html" target="_blank"><img src="https://static.igem.org/mediawiki/2011/b/b1/UCD_biosci_sponsor.jpg" width="200" height="90"></a>
-
<hp>Transformations</hp>
+
</center>
 +
 
 +
<center>
 +
<a href="http://www.genomecenter.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/1/1b/UCD_Genome_center_sponsor.jpg" width="200" height="60"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://www.cs.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/6/6b/UCD_Computer_sponsor.jpg" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://www.bme.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/4/40/UCD_BME_logo_minimal_copy.png" width="200 height="70"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://www.fishersci.com" target="_blank"><img src="https://static.igem.org/mediawiki/2011/a/a4/UCD_Fisher_Logo.gif" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://www.arcadiabio.com/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/4/46/UCD_Arcadia_sponsor.jpg
 +
" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://provost.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/8/82/UCD_Provost_sponsor.jpg
 +
" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a href="http://www.research.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/9/99/UCD_Research_sponsor.jpg" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
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<center>
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<a title="" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/25/UCD_Schultz_sponsor.jpg
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" width="200"></a>
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</div>
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</div>
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<div id="myleftbox">
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<div id="myleftbox"  class="smallbox">
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<h1>Protocols</h1>
 +
<!-- accordion starts here -->
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<div id="firstpane" class="menu_list">
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 +
  <p class="menu_head"> Registry Part Distribution Rehydration </p>
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    <div class="menu_body">
 +
<ul>
 +
<li>Add 20 µL sterile H2O to desired well in distribution plate.</li>
 +
<li>Incubate at room temperature for ~10 minutes.</li>
 +
<li>Transfer resuspension to microcentrifuge tube.</li>
 +
</ul>
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    </div>
 +
 
 +
  <p class="menu_head"> Transformations</p>
 +
    <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
•Competent Cells
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<ul>
-
<br>•DNA template
+
<li>Competent Cells</li>
-
<br>•800 ul LB
+
<li>DNA template</li>
-
<br>•LB+Antibiotic Plates
+
<li>800 µL LB</li>
 +
<li>LB+Antibiotic Plates</li>
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Thaw competent cells on ice.
+
<ul>
-
<br>•Transfer 50 uL of competent cells to chilled falcon tubes.
+
<li>Thaw competent cells on ice.</li>
-
<br>•Add 1 uL of template to cells (2.5 uL if dilute).
+
<li>Transfer 50 µL of competent cells to chilled falcon tubes.</li>
-
<br>•Incubate on ice for 30 minutes.
+
<li>Add 1 µL of template to cells (2.5 µL if dilute).</li>
-
<br>•Heat schock in 42 °C water bath for 90 seconds.
+
<li>Incubate on ice for 30 minutes.</li>
-
<br>•Immediately place back onto ice for 2 minutes.
+
<li>Heat schock in 42 °C water bath for 90 seconds.</li>
-
<br>•Add 800 uL of LB to each tube.
+
<li>Immediately place back onto ice for 2 minutes.</li>
-
<br>•Incubate at 37 °C for 1 hour.
+
<li>Add 800 µL of LB to each tube.</li>
-
<br>•Place 200 uL of the transformed cells on plates containing LB and the appropriate antibiotic.
+
<li>Incubate at 37 °C for 1 hour.</li>
-
<br>•Incubate overnight at 37 °C.
+
<li>Place 200 µL of the transformed cells on plates containing LB and the appropriate antibiotic.</li>
-
</article>
+
<li>Incubate overnight at 37 °C.</li>
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  </div>
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</ul>
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    </div>
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<br><div class="floatbox3">
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  <p class="menu_head"> Restriction Enzyme Double Digest</p>
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<article>
+
    <div class="menu_body">
-
<hp>Restriction Enzyme Double Digest</hp>
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<p>Materials</p>
<p>Materials</p>
-
•22 uL dH20
+
<ul>
-
<br>•1 uL BSA
+
<li>22 µL dH20
-
<br>•5 ul Buffer
+
<li>1 µL BSA
-
<br>•20 uL Template
+
<li>5 µLBuffer
-
<br>•1 uL Enzyme 1
+
<li>20 µL Template
-
<br>•1 uL Enzyme 2
+
<li>1 µL Enzyme 1
 +
<li>1 µL Enzyme 2
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Mix reactants, adding enzyme last.
+
<ul>
-
<br>•Place at 37 C for 3-5 hours.
+
<li>Mix reactants, adding enzyme last.
-
<br>•If not purifying or running on a gel immediately, increase to 80 C for 20 minutes to kill enzymes (some enzymes need only a 65 C heatkill, check enzyme).
+
<li>Place at 37 °C for 3-5 hours.
-
<br>•Run on a gel and extract product.
+
<li>If not purifying or running on a gel immediately, increase to 80 °C for 20 minutes to kill enzymes (some enzymes need only a 65 °C heatkill, check enzyme).
-
</article></div>
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<li>Run on a gel and extract product.
 +
</ul>
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    </div>
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<br><div class="floatbox3">
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  <p class="menu_head"> PCR (sequence dependent)</p>
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<article>
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    <div class="menu_body">
-
<hp>PCR (sequence dependent)</hp>
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<p>Materials</p>
<p>Materials</p>
-
•10 uL Q Solution
+
<ul>
-
<br>•5 uL 10x Buffer
+
<li>10 uL Q Solution
-
<br>•1.25 uL 10mM dNTPs
+
<li>5 µL 10x Buffer
-
<br>•1 uL Template
+
<li>1.25 µL 10mM dNTPs
-
<br>•1 uL Forward Primer
+
<li>1 µL Template
-
<br>•1 uL Reverse Primer
+
<li>1 µL Forward Primer
-
<br>•0.3 uL Taq
+
<li>1 µL Reverse Primer
-
<br>•0.15 uL PFU
+
<li>0.3 µL Taq
-
<br>•30 uL dH20
+
<li>0.15 µL PFU
 +
<li>30 µL dH20
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
+
<ul>
-
<br>•Run in thermal cycler.
+
<li>Mix reactants into PCR tubes. A large mix can be made and then aliquoted into PCR tubes if desired.
-
</article></div>
+
<li>Run in thermal cycler.
 +
</ul>
 +
  </div>
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<br><div class="floatbox3">
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  <p class="menu_head">PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension)</p>
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<article>
+
    <div class="menu_body">
-
<hp>SOEing</hp>
+
<p>Materials</p>
-
<br>PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension): As an important step in our project we needed to use a technique called PCR SOEing which is used to attach DNA fragments together without the many steps of digesting and ligating. Our two enzymes that break down ethylene glycol, glycoaldehyde reductase and glycoaldehyde dehydrogenase, needed to have the Eco, Xba, Spe or Pst restriction sites removed in order to biobrick the enzyme. So we had to look at the sequence of both enzymes and determine if they had any of the restriction site sequences. What we found was that glycoaldehyde reductase had one Pst restriction site and glcoaldehyde dehydrogenase had two Pst sites. </br></br>
+
<ul>
-
How SOEing works: in order to do SOEing, forward and reverse primers must be designed for where you want to attach and the whole enzyme as well. This is done by PCR reactions, and the ways we assay if the DNA fragments annealed is by doing a digest with the coordinating restriction enzyme.
+
<li>10 µL Q Solution
 +
<li>5 µL 10x Buffer
 +
<li>1.25 µL 10mM dNTPs
 +
<li>2.5 µL Forward Primer
 +
<li>2.5 µL Reverse Primer
 +
<li>0.3 µL Taq
 +
<li>0.15 µL PFU
 +
<li>Template based on concentrations determined by SOEing calculator: “Link”
 +
<li>±18.30 µL dH20 based on concentrations of template (above) by SOEing calculator: “Link again”
 +
</ul>
 +
<p>Procedure</p>
 +
<ul>
 +
<li>Mix reactants into PCR tubes.  
 +
<li>Run in thermal cycler.
 +
<li>Continue PCR SOEing of parts until completed.
 +
</ul>
 +
    </div>
-
The protocol is coming soon!
+
  <p class="menu_head"> Ligation</p>
-
</article></div>
+
    <div class="menu_body">
 +
<p>Materials</p>
 +
<ul>
 +
<li>Digested Vector
 +
<li>Digested Insert
 +
<li>Water
 +
<li>T4 DNA Ligase
 +
<li>T4 DNA Ligase Buffer
 +
</ul>
 +
<p>Procedure</p>
 +
<ul>
 +
<li>Mix these materials in the amounts determined by the reaction volume calculator <a href="https://static.igem.org/mediawiki/2011/7/73/UC_Davis_Reaction_Volume_Calculator.xls">here</a>.
 +
</ul>
 +
    </div>
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<br><div class="floatbox3">
+
  <p class="menu_head"> Ethylene Glycol Media</p>
-
<article>
+
    <div class="menu_body">
-
<hp>Ligation</hp>
+
<p>Materials</p>
<p>Materials</p>
-
•Digested Vector
+
<ul>
-
<br>•Digested Insert
+
<li>34 mM NaH<sub>2</sub>PO<sub>4</sub>
-
<br>•Water
+
<li>64 mM K<sub>2</sub>HPO<sub>4</sub>
-
<br>•T4 DNA Ligase
+
<li>20 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
-
<br>•T4 DNA Ligase Buffer
+
<li>1 µM FeSO<sub>4</sub>
 +
<li>0.1 mM MgSO<sub>4</sub>
 +
<li>10 µM CaCl<sub>2</sub>
 +
<li>30 mM Ethylene Glycol
 +
<li>30 mM Glycolate
 +
<li>0.5% wt/vol Casein Acid Hydrolysate
 +
<li>1.5% wt/vol Agar (if making solid media)
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
•Mix these materials in the amounts determined by the reaction volume calculator <a href="https://static.igem.org/mediawiki/2011/7/73/UC_Davis_Reaction_Volume_Calculator.xls">here</a>.
+
<ul>
-
</article></div>
+
<li>Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.
 +
<li>Mix together NaH<sub>2</sub>PO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, FeSO<sub>4</sub>, MgSO<sub>4</sub>, and CaCl<sub>2</sub>.
 +
<li>Titrate to pH 7 with HCl.
 +
<li>Add the glycolate and casein acid hydrolysate.
 +
<li>Mix in the ethylene glycol in a fume hood.
 +
<li>If making solid media, also mix in agar.  
 +
<li>Autoclave on an appropriate cycle.   
 +
</ul>
 +
    </div>
-
<br><div class="floatbox3">
+
  <p class="menu_head">Ethylene Glycol Toxicity Assay</p>
-
<article>
+
    <div class="menu_body">
-
<hp>Ethylene Glycol Media</hp>
+
<p>Materials</p>
<p>Materials</p>
-
•34 mM NaH<sub>2</sub>PO<sub>4</sub>
+
<ul>
-
<br>•64 mM K<sub>2</sub>HPO<sub>4</sub>
+
<li>Luria Broth Media
-
<br>•20 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
+
<li>Ethylene Glycol
-
<br>•1 µM FeSO<sub>4</sub>
+
<li>Tecan M200 Pro
-
<br>•0.1 mM MgSO<sub>4</sub>
+
<li>E. <I>coli</I>
-
<br>•10 µM CaCl<sub>2</sub>
+
</ul>
-
<br>•30 mM Ethylene Glycol
+
-
<br>•30 mM Glycolate
+
-
<br>•0.5% wt/vol Casein Acid Hydrolysate
+
-
<br>•1.5% wt/vol Agar (if making solid media)
+
<p>Procedure</p>
<p>Procedure</p>
-
•Be sure to wear a lab coat while making this media, since both glycolate and ethylene glycol are irritants.  
+
<ul>
-
<br>•Mix together NaH<sub>2</sub>PO<sub>4</sub>, K<sub>2</sub>HPO<sub>4</sub>, (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, FeSO<sub>4</sub>, MgSO<sub>4</sub>, and CaCl<sub>2</sub>.  
+
<li>Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.  
-
<br>•Titrate to pH 7 with HCl.  
+
<li>Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
-
<br>•Add the glycolate and casein acid hydrolysate.  
+
<li>Incubate at 37°C overnight on a shaker at 150 rpm.
-
<br>•Mix in the ethylene glycol in a fume hood.  
+
<li>From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.
-
<br>•If making solid media, also mix in agar.  
+
<li>Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
-
<br>•Autoclave on an appropriate cycle.  
+
<li>Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.  
-
</article></div>
+
<li>Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
 +
<li>With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.  
 +
<li>Add “blank” wells of just LB media to an entire column to serve as a control for the LB.
 +
<li>Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.  
 +
<li>Aliquot the remaining dilutions as the diagram below depicts.  
 +
<li>Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.
 +
<li>Follow the steps of your Tecan machine appropriate to the specific bacterial strain.  
 +
</ul>
 +
    </div>  
-
<br><div class="floatbox3">
+
  <p class="menu_head"> EMS (Ethyl methanesulfonate Mutagenesis)</p>
-
<article>
+
    <div class="menu_body">
-
<hp>Ethylene Glycol Toxicity Assay</hp>
+
<p>Materials</p>
<p>Materials</p>
-
• Luria Broth Media
+
<ul>
-
<br>• Ethylene Glycol
+
<li>Buffer A: Minimal medium + 0.2 M Tris (pH 7.5). per liter:
-
<br>• Tecan M200 Pro
+
<ul>
-
<br>• E. <I>coli</I>
+
<li>K<sub>2</sub>HPO<sub>4</sub>: 10.5g</li>
 +
<li>KH<sub>2</sub>PO<sub>4</sub>: 4.5g</li>
 +
<li>(NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>: 1g</li>
 +
<li>Sodium Citrate * 2H<sub>2</sub>O: 0.5g</li>
 +
<li>1M Tris (pH 7.5): 200mL Tris</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
<sub>Obtained components from “Miller,  J. H. (1972) Experiments  in Molecular Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY)” pg 432</sub>
 +
<ul>
 +
<li>EMS (Sigma)</li>
 +
<li>50mL conical Corning tubes</li>
 +
<li>15mL Falcon tubes</li>
 +
<li>LB Broth</li>
 +
<li>Ethylene Glycol Agar Plates</li>
 +
</ul>
<p>Procedure</p>
<p>Procedure</p>
-
• Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.
+
<ul>
-
<br>1. Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
+
<li>1. Prepare a 10mL liquid culture in LB in a 50mL conical tube and grow it overnight until it reaches OD 0.2.</li>
-
<br>2. Incubate at 37°C overnight on a shaker at 150 rpm.
+
<li>Chill the cells on ice and spin down the 10mL aliquots at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 10 minutes.</li>
-
<br>3. From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.  
+
<li>Wash twice with 10mL Buffer A.</li>
-
<br>4. Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
+
            <ul>
-
<br>5. Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.  
+
                <li>Pipet up and down to mix, pellet cells, decant supernatant.</li>
-
<br>6. Use 15 mL conical tubes to make dilutions of ethylene glycol with LB media (see diagram below).
+
            </ul>
-
<br>7. With a 96-well round-bottom plate, fill the outermost wells with LB media to prevent dehydration on the edges.  
+
<li>Re-suspend the pellet in 5mL of Buffer A and transfer the medium to a Falcon tube.</li>
-
<br>8. Add “blank” wells of just LB media to an entire column to serve as a control for the LB.  
+
<li>Add (35μL, 70μL, or 105μL) of EMS into each tube of re-suspended cells. </li>
-
<br>9. Include a column for 0 mM ethylene glycol and LB media as a baseline for activity without ethylene glycol.  
+
<li>Close tubes and parafilm the lid 2X.</li>
-
<br>10. Aliquot the remaining dilutions as the diagram below depicts.  
+
<li>Vortex to mix and place in a secondary container (50mL conical tube).</li>
-
<br>11. Add the appropriate amount of cells to the wells for a concentration of 0.01 OD before any of the readings.  
+
<li>Transfer to mixing platform and agitate at ~30 rpm at 37°C.</li>
-
<br>12. Follow the steps of your Tecan machine appropriate to the specific bacterial strain.  
+
<li>Withdraw samples at fixed time points.</li>
 +
<li>At each time point, take 1mL aliquots of the sample and place in a 15mL Falcon tube. Place in a secondary container (50mL Conical tube) and spin at max speed in <i>Eppendorf Centrifuge 5810 R</i> for 5 minutes. </li>
 +
<li>Discard supernatant in waste container. </li>
 +
<li>Wash twice in 5mL of Buffer A and discard supernatant in waste container.</li>
 +
<li>Re-suspend in 5mL of Buffer A and titer for viable cells.</li>
 +
<li>Add 0.5mL of mutagenized culture into 10mL of LB broth in a 50mL conical tube.</li>
 +
<li>Grow cultures overnight at 37°C and plate for mutants on EG agar plates.</li>
 +
<li>Look for viable cells</li>
 +
</ul>
-
</article></div>
+
<p>Safety: Health Hazards</p>
 +
<ul>
 +
<li>Acute toxicity (oral, dermal, inhalation), category 4 </li>
 +
<li>Skin irritation, category 2 </li>
 +
<li>Eye irritation, category 2 </li>
 +
<li>Skin sensitization, category 1 </li>
 +
<li>Specific Target Organ Toxicity – Single exposure, category 3</li>
 +
<li>LD50 – 470mg/kg</li>
 +
</ul>
 +
<p>Recommended Safety & Handling</p>
 +
<ul><li>Always work in fume hood and wear lab coat, goggles, and gloves.</li></ul>
 +
    </div>
 +
 +
<p class="menu_head">Construction of a Transposase Illumina Sequencing Library</p>
 +
    <div class="menu_body">
 +
 +
<p>Procedure</p>
 +
<ul>
 +
<li>Extract genomic DNA using any proprietary gDNA extraction kit. Once DNA is extracted, quantify your DNA and run it on an agarose gel to check DNA quality.</li>
 +
<li>Prepare Illumina Adapters for Transposase Priming, Mix the Following:
 +
      <ul><li>10µM No_PCR_Adapter_1: 10µL</li>
 +
      <li>10µM No_PCR_Adapter_2: 10µL</li>
 +
      <li>10µM Mosaic Element: 20µL</li>
 +
      <li>5M NaCl: 1.2µL</li>
 +
      <li>Incubate at 98C for 5 mins and let cool to room temperature</li></ul></li>
 +
<li>Prime Transposase
 +
      <ul><li>Transposase: 10µL</li>
 +
      <li>Annealed adapter (above): 5µL</li>
 +
      <li>Water: 15µL</li>
 +
      <li>Incubate at room temperature for 20-30 mins.</li></ul></li>
 +
<li>Tagmentation Reaction
 +
      <ul><li>Primed Transposase: 10µL (depends on the concentration of available Tn5)</li>
 +
      <li>200ng of gDNA extration: XµL</li>
 +
      <li>5x Tn5 Buffer: 3µL</li>
 +
      <li>Water: up to 15µL</li>
 +
      <li>Run multiple reactions in parallel to achieve appropriate amount of input DNA for sequencing platform</li>
 +
      <li>Incubate at 55C for 10 mins, and immediately pool reactions and clean up using a PCR clean up kit, elute with 15µL water</li></ul></li>
 +
<li>Gap Filling
 +
      <ul><li>Run a typical PCR reaction, without oligos, at 72C for 5 mins</li></ul></li>
 +
<li>Size Selection
 +
      <ul><li>Run gap filling products on 2% agarose gel, size select for the appropriate size required for your sequencing platform</li></ul></li>
 +
<li>Quality check your library
 +
      <ul><li>Run bioanalyzer to check your library quality, and qPCR to quantify your library before submission.</li></ul></li>
 +
<li>SUBMIT!</li>
 +
</ul>
</div>
</div>
-
<script>if (window.runOnloadHook) runOnloadHook();</script>
+
 
 +
<p class="menu_head">Cutinase Expression and Western Blot</p>
 +
    <div class="menu_body">
 +
 
 +
<p>Procedure</p>
 +
<ul>
 +
<li>Prepare a starter culture of MG1655 <i>E. coli</i> with pBad regulated and his-tagged LC-Cutinase along with a negative control.</li>
 +
<li>Inoculate new 5 mL cultures from the starter cultures at an OD 600 of about 0.05.</li>
 +
<li>Let the cultures grow until and OD 600 of 0.5, then inoculate two 100 mL cultures (one for the his-tagged protein and one for the negative control). </li>
 +
<li>Once these cultures are at an OD of 0.8, separate into two 50 mL cultures. Induce one at 10 uM, leave the other uninduced.</li>
 +
<li>Take 1 mL samples at different time points.</li>
 +
<li>Centrifuge samples at 5000g for 5 minutes then take off and save media.</li>
 +
<li>Wash cells with water.</li>
 +
<li>Resuspend with 300 uL of B-PER Protein Extraction Reagent and follow the instructions provided with that kit. After this, there should be samples of culture media, and soluble and insoluble whole cell lysate.</li>
 +
<li> Take 15 uL of each sample, including controls, and run on western blot.</li>
 +
 
 +
    </div>
 +
 
 +
<p class="menu_head">pNPB Assay</p>
 +
    <div class="menu_body">
 +
 
 +
<p>Materials</p>
 +
<ul>
 +
<li>pNPB buffer (should be made and used in fumehood due to acetonitriles toxicity)
 +
<ul><li>10mM pNPB in acetonitrile</li>
 +
<li>1:4:95 ratio of acetonitrile pNPB solution, 100% ethanol and 50mM Tris-HCL (pH 8)</li></ul></li>
 +
<li>LB with specific resistance</li>
 +
<li>Cell Culture</li>
 +
<li>96 well plate</li></ul>
 +
<br>
 +
<p>Protocol</p>
 +
<ul><li>First assign each well of the plate except for outer wells which should be filled with LB to prevent evaporation of medium.</li>
 +
<li>Fill wells with 95 uL of LB</li>
 +
<li>Fill wells with 5 uL of cell culture</li>
 +
<li>In fumehood, add 100 uL of bufer to each well</li>
 +
<li>Run in Tecan taking both ODs and absorbance 405 readings</li>
 +
 
 +
    </div>
 +
 
 +
<p class="menu_head">SDM (Site Directed Mutagenesis)</p>
 +
    <div class="menu_body">
 +
 
 +
<p>Materials</p>
 +
<ul>
 +
<li>Pfu turbo</li>
 +
<li>10X Pfu turbo buffer</li>
 +
<li>dNTPs (10mM)</li>
 +
<li>Forward and reverse primers (0.1ug/uL, see methods section for design tips)</li>
 +
<li>dH<sub>2</sub>0</li>
 +
<li>Dpn1</li>
 +
<li>Competent cells</li>
 +
</ul>
 +
<br>
 +
<p>Methods</p>
 +
<ul>
 +
<li>Primer Design</li>
 +
<ul>
 +
<li>Forward primer should be between 25 and 45 bases in length and contain the desired mutation in the center with correct sequences on both sides; the reverse primer is the reverse complement of this.</li>
 +
<li>Primers should have a minimum GC content of 40% and terminate in one or more C's or G's.</li>
 +
<li>Tm should be greater or equal to 78°C and can be calculated as follows:</li>
 +
<li>Tm = 81.5 + 0.41(%GC) - 675/(length in bases) - %mismatch</li>
 +
</ul></ul>
 +
<ul>
 +
<li>Reaction</li>
 +
<ul>
 +
<li>Template DNA: 1 µL</li>
 +
<li>10X Buffer: 5 µL</li>
 +
<li>Forward Primer (100 ng/µL): 1µL</li>
 +
<li>Reverse Primer (100 ng/µL): 1µL</li>
 +
<li>10mM dNTPs: 1µL</li>
 +
<li>Pfu Turbo (Stratagene): 1µL</li>
 +
<li>MilliQ H<sub>2</sub>0: 40µL</li>
 +
</ul>
 +
<li>PCR Program</li>
 +
<ul>
 +
<li>95°C for 1 minute</li>
 +
<li>95°C for 50 seconds, 60°C for 50 seconds, 68°C for 1 minute/kb of plasmid length -- repeat this step 17 times, or 18 cycles total</li>
 +
<li>68°C for 7 minutes</li>
 +
<li>4°C hold</li>
 +
<li>Following PCR - save 4µL of PCR reaction.</li>
 +
<li>To remaining 46µLs add 1µL of Dpn1 to PCR reaction (PCR cleanup is not necessary). Incubate at 37°C for 1-2 hours to digest parental DNA. Run 5µL of the digested reaction on a gel and compare to the undigested parental plasmid - there should be some difference in band pattern. Transform into competent cells (PCR cleanup is not necessary).</li>
 +
</ul>
 +
 
 +
<br>
 +
<li>Protocol Adapted From <a href="http://mcmanuslab.ucsf.edu/protocol/site-directed-mutagenesis-stratagene-protocol">Here</a>
 +
</ul>
 +
    </div>
 +
 
 +
 
 +
 
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