Difference between revisions of "Team:UPMC-Paris/Experiments"

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{{UPMC-Paris}}
 
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<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
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<h5>What should this page contain?</h5>
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<li> Protocols </li>
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<li> Experiments </li>
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<li>Documentation of the development of your project </li>
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<!--- THIS IS WHERE THE HTML BEGINS --->
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<head>
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<meta name="viewport" content="width=device-width, initial-scale=1">
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</head>
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<div>
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<div class="collapsable_menu_control"> MENU ▤ </div>
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<ul id="accordion" class="accordion">
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<li class="menu_item"> <div class="icon plus"></div> Competent Cells
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<ul class="submenu">
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<li>  
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<a href="">
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<div>
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<h>Preparation of Bacillus subtilis competent cells</h>
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<ol><li>Streak out the strain to be made competent on an LB agar plate as a large patch and incubate overnight at 30°C</li>
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<li>The following morning scrape the cell growth off the plate and use to inoculate fresh, pre-warmed, SpC medium (20 ml) to give an OD600 reading of about 0.5. </li>
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<li>Incubate the culture at 37°C with vigorous aeration and take periodic OD readings (OD600) to assess cell growth.</li>
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<li>When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 min) inoculate 200 ml of pre-warmed, SpII medium with 2 ml of stationary-phase culture and continue incubation at 37°C with slower aeration </li>
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<li>After 90 min incubation, pellet the cells by centrifugation (8,000 g, 5min) at room temperature.</li>
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<li>Carefully decant the supernatant into a sterile container and save.</li>
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<li>Gently resuspended the cell pellet in 18 ml of the saved supernatant and add 2 ml of sterile glycerol; mix gently </li>
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<li>Aliquot the competent cell (0.5 ml) in sterile tubes, freeze rapidly in liquid nitrogen or a dry-iced/ethanol bath or ice/isopropanol bath and store -70.</li></ol>
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</div></a></li></ul></li>
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<li class="menu_item"> <div class="icon plus"></div> Transformation
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<ul class="submenu">
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<li>
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<a href="">
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<div>
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<h>Cells Preparation :</h>
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<ol><li>Thaw competent cells rapidly by immersing frozen tubes in a 37°C water bath</li>
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<li>Immediately, add one volume of SpII + EGTA to the Thawed cells; mix gently </li>
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<li>In a sterile test tube add competent cell (0.2~0.5 ml) to the DNA solution (<0.1 ml) and incubate in a roller drum at 37.</li>
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<li>Dilute the transformed cells as appropriate in T base containing 0.5% glucose and plate immediately onto selective media. </li></ol>
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<p align="center"><img src="https://static.igem.org/mediawiki/igem.org/d/d3/Transfo_pic_1.png" width="400px"/></p>
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</div></a>
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<a href=""><div>
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<h>Digestion :</h>
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<ol><li>Select restriction enzymes to digest your plasmid. Determine an appropriate reaction buffer by reading the instructions for your enzyme. </li>
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<p>In a 1.5mL tube combine the following:</p>
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<ul><li>DNA </li>
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<li>Restriction Enzyme(s)</li>
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<li>Buffer </li>
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<li>dH2O up to total volume</li> </ul>
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<p>Mix gently by pipetting. </p>
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<li>Incubate tube at appropriate temperature (usually 37°C) for 1 hour. </li>
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<li>Always follow the manufacturer’s instructions. </li>
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<li>To visualize the results of your digest, conduct gel electrophoresis</li></ol>
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</div></a>
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<a href=""><div>
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<h>Vector Preparation :</h>
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<p>Combine the following in a PCR or Eppendorf tube:</p>
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<ul><li>25ng Vector DNA</li>
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<li>75ng Insert DNA</li>
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<li>Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)</li>
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<li>0.5-1μL T4 DNA Ligase</li>
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<li>H20 to a total of 10μL</li></ul>
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<p>Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s instructions).</p>
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</div></a></li></ul></li>
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<li class="menu_item"> <div class="icon plus"></div> Deletion
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<ul class="submenu">
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<li>
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<a href="">
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<div>
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<p>Deletion Step 1 :</p>
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<img src="aaa" width="500px"/>
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<p>Deletion Step 2 :</p>
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<img src="aaa" width="500px"/>
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<p>Deletion Step 3 :</p>
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<img src="aaa" width="500px"/></div></a></li></ul></li>
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<li class="menu_item"> <div class="icon plus"></div> Other
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<ul class="submenu">
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<li>
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<a href="">
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<div>
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<p>Whatever Step 1 :</p>
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<img src="aaa" width="50px"/>
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<p>Whatever Step 2 :</p>
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<img src="aaa" width="50px"/></div></a>
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<a href="">
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<div>
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<p>Un autre truc Step 1 :</p>
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<img src="aaa" width="50px"/>
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<p>Un autre truc Step 2 :</p>
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<img src="aaa" width="50px"/></div></a></li></ul></li>
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 +
</div>
  
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<div class="column half_size">
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<h5>Inspiration</h5>
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<ul>
 +
<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
 +
<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
 
</ul>
 
</ul>
 
 
</div>
 
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</div>
<h1 id="team_name">  </h1>
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<h4 id="page_name">  </h4>
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Revision as of 15:41, 31 July 2016