Team:Alberta/Protocols

From 2013.igem.org

(Difference between revisions)
m
 
(19 intermediate revisions not shown)
Line 19: Line 19:
         height:37px;
         height:37px;
         margin-top:-17px;
         margin-top:-17px;
-
         margin-left:-110px;
+
         margin-left:-95px;
       }
       }
     }
     }
Line 55: Line 55:
     }
     }
     .sidebar {
     .sidebar {
-
       position:fixed;
+
       position: fixed;
-
       top:190px;
+
       top: 157px;
-
       float:left;
+
       float: left;
-
       z-index:3;
+
       z-index: 3;
-
       padding: 0 0px 0px 40px;
+
       padding: 10px 10px 10px 10px;
 +
      width: 230px;
 +
    }
 +
    .sidebar_block {
 +
      background-color:white;
 +
      margin:1px;
 +
      border:1px grey solid;
 +
      padding:5px 10px 5px 10px;
 +
      border-radius:5px;
 +
      box-shadow: 0px 0px 20px #444444;
     }
     }
     .main {
     .main {
Line 78: Line 87:
       position:fixed;
       position:fixed;
       margin-left:650px;
       margin-left:650px;
-
       margin-top:-100px;
+
       margin-top:-95px;
       width: 150px;
       width: 150px;
 +
    }
 +
    .igem-logo img {
 +
      width:210px;
     }
     }
     .igem-bar {
     .igem-bar {
Line 371: Line 383:
       box-shadow: 0px 0px 30px #000;
       box-shadow: 0px 0px 30px #000;
        
        
 +
    }
 +
    a.anchor{
 +
      display: block;
 +
      position: relative;
 +
      top: -180px;
 +
      visibility: hidden;
     }
     }
   </style>
   </style>
Line 377: Line 395:
     <div class="titlebar">
     <div class="titlebar">
<br>
<br>
-
      <h3><font color="#A80000"><font size="6">The Littlest Mapmaker</font></font></h3>
+
<img src="/wiki/images/4/41/Ab_mapmen_title.png" width="600" height="95"></img><img src="/wiki/images/a/a5/2013-igem-logo.png" width="198" height="95"></img>
-
      <h4><i><font size="2">"Exploration into the world of <font color="#A80000">DNA Computing</font>"<br/>
+
-
        Team Alberta: University of Alberta</font></i></h4>
+
-
      <a href="http://www.ualberta.ca" class="ualberta-logo"><img src="/wiki/images/b/b3/Ualberta-logo.png" alt="University of Alberta"></img></a>
+
-
      <a href="https://2013.igem.org" class="igem-logo"><img src="/wiki/images/a/ad/IGem-logo.png" alt="iGem Main Page"></img></a>
+
     </div>
     </div>
     <div class="bin">
     <div class="bin">
       <ul class="navbar">
       <ul class="navbar">
         <li><a  href="/Team:Alberta">Home</a></li>
         <li><a  href="/Team:Alberta">Home</a></li>
-
         <li><a id="project" href="#">Project</a>
+
         <li><i class="nav-icon"></i><a id="project" href="#" class="active">Project</a>
           <ul>
           <ul>
             <li><a href="/Team:Alberta/Background">Background</a></li>
             <li><a href="/Team:Alberta/Background">Background</a></li>
             <li><a href="/Team:Alberta/Overview">Overview</a></li>
             <li><a href="/Team:Alberta/Overview">Overview</a></li>
-
            <li><a href="/Team:Alberta/FutureDevelopment">Future Development</a></li>
 
-
          </ul>
 
-
        </li>
 
-
        <li><i class="nav-icon"></i><a id="notebook" href="#" class="active">Notebook</a>
 
-
          <ul>
 
             <li><a href="/Team:Alberta/Results">Results</a></li>
             <li><a href="/Team:Alberta/Results">Results</a></li>
             <li><a href="/Team:Alberta/Protocols" class="active">Protocols</a></li>
             <li><a href="/Team:Alberta/Protocols" class="active">Protocols</a></li>
-
             <li><a href="/Team:Alberta/Parts">BioBricks</a></li>
+
             <li><a href="/Team:Alberta/Parts">Submitted Parts</a></li>
 +
            <li><a href="/Team:Alberta/Accomplishments">Accomplishments</a></li>
           </ul>
           </ul>
         </li>
         </li>
Line 404: Line 414:
             <li><a href="/Team:Alberta/Team">Roster</a></li>
             <li><a href="/Team:Alberta/Team">Roster</a></li>
             <li><a href="https://igem.org/Team.cgi?year=2013&team_name=Alberta">Official Profile</a></li>
             <li><a href="https://igem.org/Team.cgi?year=2013&team_name=Alberta">Official Profile</a></li>
-
             <li><a href="/Team:Alberta/Attributions">Sponsors</a></li>
+
             <li><a href="/Team:Alberta/Sponsors">Sponsors</a></li>
 +
            <li><a href="/Team:Alberta/Attributions">Attributions</a></li>
           </ul>
           </ul>
         </li>
         </li>
Line 423: Line 434:
       </ul>
       </ul>
       <div class="sidebar">
       <div class="sidebar">
-
         <div href class="tooltip"><img src="/wiki/images/7/7d/SideChar.png"></img>  
+
         <div class="sidebar_block">
-
           <span class="saying">Welcome to the Team Alberta Wiki!
+
          <h5>Places</h5>
-
             <div id="box" style="width: 450px; padding: 5px; border: 3px solid #000; background-color: #000000;">
+
          <a href="#Top"><p>Top</p></a>
-
             <div id="template" style="text-align: center; font-weight: bold; font-size: large; color: #f6f6f6; padding: 5px;">
+
          <a href="#Parts"><p>The Parts</p></a>
-
              For visitors: this site is currently under construction. Please contact our Student Liason, Dawson at                  daocun@ualberta.ca, for more information on our current project and how to support us!
+
           <a href="#Build"><p>Building the Plasmids</p></a>
-
             </div>
+
          <h5>Project Sections</h5>
-
             </div>
+
            <a href="/Team:Alberta/Background"><p>Background</p></a>
-
          </span>
+
             <a href="/Team:Alberta/Overview"><p>Overview</p></a>
 +
            <a href="/Team:Alberta/Results"><p>Results</p></a>
 +
             <a href="/Team:Alberta/Protocols" class="active"><p>Protocols</p></a>
 +
             <a href="/Team:Alberta/Parts"><p>Submitted Parts</p></a>
 +
             <a href="/Team:Alberta/Accomplishments"><p>Accomplishments</p></a>
         </div>
         </div>
       </div>
       </div>
       <div class="main">
       <div class="main">
 +
      <a class="anchor" id="Top"></a>
       <div class="block">
       <div class="block">
         <p class="content-title">Protocols</p>
         <p class="content-title">Protocols</p>
       </div>
       </div>
 +
<a class="anchor" id="Parts"></a>
 +
      <div class="block">
 +
        <h2>The Parts</h2>
 +
        <p>The 6 genes that were required to assemble various routes were obtained by PCR from plasmids kindly provided by Genomikon Inc. Each gene (AmpR, KanR, ClrR, GFP, RFP and aCP) exist as self contained cassettes that are flanked by BsaI sites. Parts were then generated according to the schematic shown below:</p>
 +
<img src="/wiki/images/c/c5/Making_parts_678x586.png" align="middle" style="width:100%;">
 +
      </div>
 +
      <a class="anchor" id="Build"></a>
       <div class="block">
       <div class="block">
-
         <p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut           
+
        <h2>Building the Plasmids</h2>
-
          laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation
+
         <p>Our plasmid assembly system relies upon the achievements of previous Team Alberta iGEM entries, 2009’s BioBytes and 2010’s Genomikon assembly methods. This process begins with origins of replication anchored at one end to magnetic beads in a suspension within a reaction microfuge tube. The anchored strands have a single, free-floating sticky end, onto which successive genes are ligated. Once the new gene has been ligated on, we use a magnet to hold the beads (along with the anchored DNA) inside the reaction tube, while washing away the rest of the reaction, including the enzyme, buffers, and any non-ligated DNA that remains. The beads are then resuspended in a new reaction mixture, containing the next ligation step.</p>
-
          ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in  
+
<img src="/wiki/images/b/b4/2013Alberta-Poster2.jpg" align="middle" style="width:100%;">
-
          hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero         
+
<p>Once all of the genes have been ligated, a tail-piece that complements the original bead-anchor DNA sequence is added, so that the finished product can be unbound from the beads and will close upon itself to form the circular plasmid. In this fashion, a four-gene, roughly 5000-base-pair plasmid is assembled in as little as an afternoon, cheaply and easily.</p>
-
          eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te
+
-
          feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id
+
-
          quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui
+
-
          facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius.
+
-
          Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam
+
-
          littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula
+
-
          quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fiant sollemnes in  
+
-
          futurum.</p>
+
       </div>
       </div>
       </div>
       </div>

Latest revision as of 03:46, 29 October 2013


Protocols

The Parts

The 6 genes that were required to assemble various routes were obtained by PCR from plasmids kindly provided by Genomikon Inc. Each gene (AmpR, KanR, ClrR, GFP, RFP and aCP) exist as self contained cassettes that are flanked by BsaI sites. Parts were then generated according to the schematic shown below:

Building the Plasmids

Our plasmid assembly system relies upon the achievements of previous Team Alberta iGEM entries, 2009’s BioBytes and 2010’s Genomikon assembly methods. This process begins with origins of replication anchored at one end to magnetic beads in a suspension within a reaction microfuge tube. The anchored strands have a single, free-floating sticky end, onto which successive genes are ligated. Once the new gene has been ligated on, we use a magnet to hold the beads (along with the anchored DNA) inside the reaction tube, while washing away the rest of the reaction, including the enzyme, buffers, and any non-ligated DNA that remains. The beads are then resuspended in a new reaction mixture, containing the next ligation step.

Once all of the genes have been ligated, a tail-piece that complements the original bead-anchor DNA sequence is added, so that the finished product can be unbound from the beads and will close upon itself to form the circular plasmid. In this fashion, a four-gene, roughly 5000-base-pair plasmid is assembled in as little as an afternoon, cheaply and easily.