Team:UC Davis/Notebook/Protocols

From 2012.igem.org

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 +
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 +
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#sponsorbox .sponsorfloat p
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Line 287: Line 801:
font-weight:bold;
font-weight:bold;
font-size: 40px;
font-size: 40px;
-
color: #004b85;;
+
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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/notebook_banner.jpg" width="850" height="269">
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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.
+
</center>
-
</article>
+
-
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+
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<br>
+
<center>
-
  <div class="floatbox3">
+
<a href="http://engineering.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2011/f/f6/UCD_CoE.png" width="200" height="40"></a>
-
<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>
 +
<a href="http://ucomm.ucdavis.edu/" target="_blank"><img src="https://static.igem.org/mediawiki/2012/b/b4/UCD_Communications_sponsor.jpg" width="200"></a>
 +
</center>
 +
 
 +
<center>
 +
<a title="" target="_blank"><img src="https://static.igem.org/mediawiki/2012/2/25/UCD_Schultz_sponsor.jpg
 +
" width="200"></a>
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</center>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div id="myleftbox">
 +
 
 +
<div id="myleftbox"  class="smallbox">
 +
<h1>Protocols</h1>
 +
<!-- accordion starts here -->
 +
<div id="firstpane" class="menu_list">
 +
 
 +
  <p class="menu_head"> Registry Part Distribution Rehydration </p>
 +
    <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>
 +
    </div>
 +
 
 +
  <p class="menu_head"> Transformations</p>
 +
    <div class="menu_body">
<p>Materials</p>
<p>Materials</p>
-
•Competent Cells
+
<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>
-
  </div>
+
</ul>
 +
    </div>
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<br><div class="floatbox3">
+
  <p class="menu_head"> Restriction Enzyme Double Digest</p>
-
<article>
+
    <div class="menu_body">
-
<hp>Restriction Enzyme Double Digest</hp>
+
<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>
+
<li>Run on a gel and extract product.
 +
</ul>
 +
    </div>
-
<br><div class="floatbox3">
+
  <p class="menu_head"> PCR (sequence dependent)</p>
-
<article>
+
    <div class="menu_body">
-
<hp>PCR (sequence dependent)</hp>
+
<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>
-
<br><div class="floatbox3">
+
  <p class="menu_head">PCR SOEing (Polymerase Chain Reaction Splicing by Overlapping Extension)</p>
-
<article>
+
    <div class="menu_body">
-
<hp>SOEing</hp>
+
<p>Materials</p>
-
<br>Coming soon.
+
<ul>
-
</article></div>
+
<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>
-
<br><div class="floatbox3">
+
  <p class="menu_head"> Ligation</p>
-
<article>
+
    <div class="menu_body">
-
<hp>Ligation</hp>
+
<p>Materials</p>
<p>Materials</p>
-
•Digested Vector
+
<ul>
-
<br>•Digested Insert
+
<li>Digested Vector
-
<br>•Water
+
<li>Digested Insert
-
<br>•T4 DNA Ligase
+
<li>Water
-
<br>•T4 DNA Ligase Buffer
+
<li>T4 DNA Ligase
 +
<li>T4 DNA Ligase Buffer
 +
</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>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>
-
<br><div class="floatbox3">
+
  <p class="menu_head"> Ethylene Glycol Media</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>34 mM NaH<sub>2</sub>PO<sub>4</sub>
-
<br>•20 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
+
<li>64 mM K<sub>2</sub>HPO<sub>4</sub>
-
<br>•1 µM FeSO<sub>4</sub>
+
<li>20 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
-
<br>•0.1 mM MgSO<sub>4</sub>
+
<li>1 µM FeSO<sub>4</sub>
-
<br>•10 µM CaCl<sub>2</sub>
+
<li>0.1 mM MgSO<sub>4</sub>
-
<br>•30 mM Ethylene Glycol
+
<li>10 µM CaCl<sub>2</sub>
-
<br>•30 mM Glycolate
+
<li>30 mM Ethylene Glycol
-
<br>•0.5% wt/vol Casein Acid Hydrolysate
+
<li>30 mM Glycolate
-
<br>•1.5% wt/vol Agar (if making solid media)
+
<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>
-
•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 this media, since both glycolate and ethylene glycol are irritants.  
-
<br>•Titrate to pH 7 with HCl.  
+
<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>.  
-
<br>•Add the glycolate and casein acid hydrolysate.  
+
<li>Titrate to pH 7 with HCl.  
-
<br>•Mix in the ethylene glycol in a fume hood.  
+
<li>Add the glycolate and casein acid hydrolysate.  
-
<br>•If making solid media, also mix in agar.  
+
<li>Mix in the ethylene glycol in a fume hood.  
-
<br>•Autoclave on an appropriate cycle.  
+
<li>If making solid media, also mix in agar.  
-
</article></div>
+
<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 Toxicity Assay</hp>
+
<p>Materials</p>
<p>Materials</p>
-
• Luria Broth Media
+
<ul>
-
<br>• Ethylene Glycol
+
<li>Luria Broth Media
-
<br>• Tecan M200 Pro
+
<li>Ethylene Glycol
-
<br>• E. <I>coli</I>
+
<li>Tecan M200 Pro
 +
<li>E. <I>coli</I>
 +
</ul>
 +
<p>Procedure</p>
 +
<ul>
 +
<li>Be sure to wear a lab coat while making dilutions and aliquots, since ethylene glycol is an irritant.
 +
<li>Inoculate bacterial colony in 5 mL of luria broth (LB) media in a 15 mL falcon tube to create a starter culture.
 +
<li>Incubate at 37°C overnight on a shaker at 150 rpm.
 +
<li>From the incubated starter culture, take 20 µL and add it into 3 mL of LB media.
 +
<li>Incubate at 37°C for 2-3 hours on a shaker at 150 rpm.
 +
<li>Measure the optical density (OD) reading every thirty minutes after two hours to reach an ideal range of 0.5 to 0.7.
 +
<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>
 +
 
 +
  <p class="menu_head"> EMS (Ethyl methanesulfonate Mutagenesis)</p>
 +
    <div class="menu_body">
 +
<p>Materials</p>
 +
<ul>
 +
<li>Buffer A: Minimal medium + 0.2 M Tris (pH 7.5). per liter:
 +
<ul>
 +
<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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