Difference between revisions of "Team:NTNU Trondheim/Protocols"

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<div class="row">
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<ul class="side-nav" style="margin-left:15px">
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<!-- -------------------Month Filters------------------- -->
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<li><h6> Jump to: </h6></li>
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<!--li class="nb-month">
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<div style="top:67px;margin-left:-6px">Spring</div>
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<li id="weekA" class="active" onclick="weekFilter(this)">
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<a class="nb-week" href="#A">January</a>
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<a class="nb-week" href="#B">February</a>
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<a class="nb-week" href="#C">March</a>
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<a class="nb-week" href="#D">April</a>
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<a class="nb-week" href="#E">May</a>
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<li class="nb-month">
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<div style="top:55px">Media</div>
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</li>
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<li id="week1" onclick="weekFilter(this)">
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<a class="nb-week" href="#1">Lysogeny Broth (LB)</a>
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</li>
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<li id="week2" onclick="weekFilter(this)">
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<a class="nb-week" href="#2">SOC medium</a>
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</li>
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<li id="week3" onclick="weekFilter(this)">
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<a class="nb-week" href="#3">yB medium</a>
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</li>
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<li id="week4" onclick="weekFilter(this)">
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<a class="nb-week" href="#4"><i>Synechocystis</i> medium</a>
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<li id="week5" onclick="weekFilter(this)">
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<a class="nb-week" href="#5"></a>
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<li class="nb-month">
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<div style="top:140px;margin-left:-18px">Techniques</div>
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</li>
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<li id="week6" onclick="weekFilter(this)">
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<a class="nb-week" href="#6">Gibson assembly</a>
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</li>
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<li id="week7" onclick="weekFilter(this)">
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<a class="nb-week" href="#7">DNA isolation and cleaning</a>
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</li>
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<li id="week8" onclick="weekFilter(this)">
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<a class="nb-week" href="#8">DNA digestion</a>
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</li>
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<li id="week9" onclick="weekFilter(this)">
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<a class="nb-week" href="#9">PCR</a>
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</li>
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<li id="week10" onclick="weekFilter(this)">
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<a class="nb-week" href="#10">NanoDrop</a>
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</li>
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<li id="week11" onclick="weekFilter(this)">
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<a class="nb-week" href="#11">3A assembly</a>
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</li>
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<li id="week12" onclick="weekFilter(this)">
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<a class="nb-week" href="#12">Ligation</a>
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</li>
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<li id="week13" onclick="weekFilter(this)">
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<a class="nb-week" href="#13">Transformation (<i>Escherichia coli</i>)</a>
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</li>
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<li id="week14" onclick="weekFilter(this)">
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<a class="nb-week" href="#14">Transformation (<i>Synechocystis sp. PCC 6803</i>)</a>
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</li>
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<li class="nb-month">
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<div style="top:52px;margin-left:-9px">Plasmids</div>
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</li>
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<li id="week16" onclick="weekFilter(this)">
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<a class="nb-week" href="#16">Right flank</a>
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</li>
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<li id="week17" onclick="weekFilter(this)">
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<a class="nb-week" href="#17">Left flank</a>
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</li>
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<li id="week18" onclick="weekFilter(this)">
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<a class="nb-week" href="#18">Kanamycin resistance</a>
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</li>
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<li id="week19" onclick="weekFilter(this)">
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<a class="nb-week" href="#19">Lac promoter</a>
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</li>
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<li id="week20" onclick="weekFilter(this)">
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<a class="nb-week" href="#20">Glucose oxidase</a>
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</li>
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<li id="week21" onclick="weekFilter(this)">
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<a class="nb-week" href="#21">Left flank + Kan + Right flank</a>
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</li>
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<li class="nb-month">
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<div style="top:55px;margin-left:-29px">Calculations</div>
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</li>
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<li id="week24" onclick="weekFilter(this)">
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<a class="nb-week" href="#24">DNA concentration</a>
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</li>
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</ul>
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</div>
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<div class="ten columns team-bios-container">
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<div class="row" id="ugd-members">
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<div class="twelve columns">
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<h2 class="centered">Protocols</h2>
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</div>
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</div>
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<!-- -------------------Subteam Filters------------------- -->
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<div class="row last-ele" style="padding-bottom:0px">
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<h4 style="margin-left:15px;text-align:left">Filter by subteam:
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<div id="nb-all" class="nb-all" onclick="showAll()">show all categories</div>
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<div id="nb-alltech" class="nb-all-i" onclick="showAllTech()" style="margin-right:15px">show technical details</div>
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</h4>
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</div>
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<div class="row last-ele nb-labels" style="padding-bottom:0px">
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<div style="opacity:0.00;width:65px">"_"</div>
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<div>Media</div>
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<div>Techniques</div>
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<div>Plasmids</div>
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<div>Calculations</div>
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</div>
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<div class="row">
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<!-- PLACEHOLDER -->    <div class="two columns nb-filter" style="opacity:0.00;width:65px">
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<div id="media-only" class="nb-only" onclick="onlyFilter(this)">only</div>
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<div class="two columns nb-filter">
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<div class="filter-container" onclick="filter(this)" style="background-image:url(https://static.igem.org/mediawiki/2014/9/94/NTNU_techniques_off.png);height=100px;width=100px">
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<img id="techniques" src="https://static.igem.org/mediawiki/2014/b/be/NTNU_techniques_on.png" height="100px" width="100px">
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<div id="techniques-only" class="nb-only" onclick="onlyFilter(this)">only</div>
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</div>
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<div class="two columns nb-filter">
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<div class="filter-container" onclick="filter(this)" style="background-image:url(https://static.igem.org/mediawiki/2014/f/fc/NTNU_plasmids_off.png)">
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<img id="plas" src="https://static.igem.org/mediawiki/2014/a/ad/NTNU_plasmids_om.png">
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</div>
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<div id="plas-only" class="nb-only" onclick="onlyFilter(this)">only</div>
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</div>
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<div class="two columns nb-filter">
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<div class="filter-container" onclick="filter(this)" style="background-image:url(https://static.igem.org/mediawiki/2014/b/b8/NTNU_calculations_off.png);width=100px;height=100px">
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<img id="calc" src="https://static.igem.org/mediawiki/2014/b/bb/NTNU_calculations_on.png";width="100px" height="100px">
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<div id="calc-only" class="nb-only" onclick="onlyFilter(this)">only</div>
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</div>
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<!-- PLACEHOLDER -->    <div class="two columns nb-filter" style="opacity:0.00;width:65px">
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</div>
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<!-- -------------------Protocol entries------------------- -->
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<div id="week1entry" class="nb-week" style="display: block">
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<div class="twelve columns">
 +
<h3 class="centered">Lysogeny Broth (LB)</h3>
 +
                                       
 +
 
 +
</p>
 +
 
 +
<div class="entry pc-media">
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<div>
 +
<h6>Recipe
 +
<div class="nb-onetech-i nohilite">Antibiotic additions</div>
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</h6>
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<div class="nb-tech">
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<div class="AB-table">
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<table>
 +
<tr>
 +
<th> Antibiotic</th> <th> Stock concentration</th> <th>Final concentration</th> <th>Dillution factor</th> <th>Solvent</th> <th>Storage temperature</th>
 +
</tr>
 +
<tr>
 +
<td>Ampicillin</td> <td>50 mg / mL</td> <td>50 &mu;g / mL</td> <td>1000</td> <td>Filter sterilized H<sub>2</sub>O</td> <td> 4 &deg;C</td>
 +
</tr>
 +
<tr>
 +
<td>Chloramphenicol</td><td>30 mg / mL</td><td>30 &mu;g / mL</td> <td>1000</td><td>Ethanol</td><td>-20 &deg;C</td>
 +
</tr>
 +
<tr>
 +
<td>Kanamycin</td><td>50 mg / mL</td><td>30 &mu;g / mL</td><td>1000</td><td>Filter sterilized H<sub>2</sub>O</td><td>4 &deg;C</td>
 +
</tr>
 +
<tr>
 +
<td>Spectinomycin</td><td>50 mg / mL</td><td>50 &mu;g / mL</td><td>1000</td><td>Filter sterilized H<sub>2</sub>O</td><td>4 &deg;C</td>
 +
</tr>
 +
</table>
 +
</div>
 +
</div>
 +
<p>
 +
Ingredients: <br> <ul> <li>Tryptone (10g)</li>  <li>NaCl (10g)</li> <li>Yeast Extract (5g)</li> </ul> <br> <p> <ol> <li>Fill with 1 L of distilled / filtered H<sub>2</sub>O.</li> <li>Autoclave at 121 &deg;C for 20 minutes. </li> <li>Add antibiotics if needed, after the medium has cooled down.</li>
 +
</ol></p>
 +
</p>
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</div>
 +
</div>
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<p>
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</div>
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</div>
 +
 
 +
<div id="week2entry" class="nb-week" style="display: block">
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<div class="twelve columns">
 +
<h3 class="centered">SOC medium</h3>
 +
</p>
 +
<div class="entry pc-media">
 +
<div>
 +
<h6>Recipe
 +
<div class="nb-onetech-i nohilite">show technical details</div>
 +
</h6>
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <p> Ingredients (1L): <br> <ul> <li>Bactotryptone (20 g)</li>  <li>Yeast extract (5g)</li> <li>NaCl (0.584g)</li> <li>KCl (0.186g)</li> <li>Agar (20g ONLY IF MAKING PLATES)</li> </ul> <br> <p> <ol> <li>Fill with 0.5 L of distilled / filtered H<sub>2</sub>O.</li> <li>Autoclave at 121 &deg;C for 20 minutes. </li>  <li>Add 10 mL 1M MgCl<sub>2</sub>, 10 mL MgSO<sub>4</sub> and 20 mL 1M glucose (all should be sterile) prior to use </li> 
 +
</ol>
 +
</p>
 +
</p>
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</div>
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</div>
 +
 
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</div>
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</div>
 +
 
 +
 
 +
<div id="week3entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">yB medium</h3>
 +
</p>
 +
<div class="entry pc-media">
 +
<div>
 +
<h6>Recipe
 +
 +
</h6>
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> Ingredients: <br> <ul> <li>Yeast extract (2.5g)</li>  <li>Bactotryptone (10g)</li> <li>KCL (0.38g)</li> </ul> <br> <p> <ol> <li>Fill with 0.5 L of distilled / filtered H<sub>2</sub>O.</li> <li>Add KOH until the pH is 7.4, then autoclave at 121 &deg;C for 20 minutes. </li>  <li>Add 17 mL sterile 1M MgSO4 </li> 
 +
</ol>
 +
</p>
 +
</p>
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</div>
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</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div id="week4entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Synechocystis medium</h3>
 +
</p>
 +
<div class="entry pc-media">
 +
<div>
 +
<h6>Recipe
 +
 +
</h6>
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> To grow Synechocystis cultures, BG-11 medium was made according to the recipe found on the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-physiological/bg-11-media">PhotoSynLab wiki</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
 
 +
<div id="week6entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Gibson Assembly</h3>
 +
<div class="entry pc-techniques">
 +
<div>
 +
<h6>
 +
</h6>
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p>
 +
The Gibson assembly protocol can be found at <a href="https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510">New England Biolabs</a>.
 +
</p>
 +
</div>
 +
</div>
 +
</div>
 +
</div>
 +
 
 +
 
 +
 
 +
<div id="week7entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">DNA isolation and cleaning</h3>
 +
</p>
 +
<div class="entry pc-techniques">
 +
<div>
 +
 +
 
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> For plasmid isolation, the Promega Wizard Plus SV Minipreps DNA Purification System A1460 Miniprep protocol was used. For PCR product isolation, the  QIAquick PCR Purification kit was used.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div id="week8entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">DNA digestion</h3>
 +
</p>
 +
<div class="entry pc-techniques">
 +
<div>
 +
 +
 
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p>
 +
 
 +
DNA digests for both ligation and verification used the protocol in the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/dna-ligation/restriction-enzyme-digest">PhotoSynLab wiki</a>.
 +
 
 +
 
 +
 
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
 
 +
<div id="week9entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">PCR</h3>
 +
</p>
 +
<div class="entry pc-techniques">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> The touchdown PCR procedure detailed on the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/pcr-touchdown-pcr">PhotoSynLab wiki</a> was used to amplify DNA.
 +
</p>
 +
</div>
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</div>
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</div>
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</div>
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 +
 
 +
<div id="week10entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Nanodrop</h3>
 +
</p>
 +
<div class="entry pc-techniques">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <a href="http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf">A NanoDrop ND-1000 Spectrophotometer</a> was used to determine DNA concentrations.
 +
</p>
 +
</div>
 +
</div>
 +
 
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</div>
 +
</div>
 +
 
 +
<div id="week11entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">3A assembly</h3>
 +
</p>
 +
<div class="entry pc-techniques">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> 3A assembly was conducted according to the  <a href="http://parts.igem.org/Help:Protocol/3A_Assembly">iGEM 3A assembly protocol</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
 
 +
<div id="week12entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Ligation</h3>
 +
 
 +
<div class="entry pc-techniques">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> Ligation was performed according to the protocol outlined on the  <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/pcr-touchdown-pcr">PhotoSynLab wiki</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
 
 +
<div id="week13entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Transformation (Escherichia coli)</h3>
 +
</p>
 +
<div class="entry pc-techniques">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> The protocols used for preparing competent DH5a cells, as well as the heat-shock transformation procedure employed can be found at the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/heat-shock-transformation-2">PhotoSynLab wiki</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div id="week14entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Transformation (Synechocystis sp. PCC 6803)</h3>
 +
</p>
 +
<div class="entry pc-techniques">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> The transformation procedure for transforming Synechocystis can be found at the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/synechocystis-transformation-r-hill-method">PhotoSynLab wiki</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div id="week16entry" class="nb-week" style="display: block">
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<div class="twelve columns">
 +
<h3 class="centered">Right flank</h3>
 +
 
 +
<div class="entry pc-plas">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <a href="http://parts.igem.org/Part:BBa_K1424001">BBa_K1424001</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div id="week17entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Left flank</h3>
 +
 
 +
<div class="entry pc-plas">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <a href="http://parts.igem.org/Part:BBa_K1424000">BBa_K1424000</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div id="week18entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Kanamycin resistance</h3>
 +
 
 +
<div class="entry pc-plas">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <a href="http://parts.igem.org/Part:BBa_K1424003">BBa_K1424003</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
 +
</div>
 +
 
 +
 
 +
<div id="week19entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Lac promotor</h3>
 +
 
 +
<div class="entry pc-plas">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <a href="http://parts.igem.org/Part:BBa_K1424002">BBa_K1424002</a>.
 +
</p>
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</div>
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</div>
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</div>
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<div class="twelve columns">
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<h3 class="centered">Glucose oxidase</h3>
 +
 
 +
<div class="entry pc-plas">
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<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <a href="http://parts.igem.org/Part:BBa_K1424004">BBa_K1424004</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
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</div>
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<div id="week21entry" class="nb-week" style="display: block">
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<div class="twelve columns">
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<h3 class="centered">Left flank + Kanamycin resistance + Right flank</h3>
 +
 
 +
<div class="entry pc-plas">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <a href="http://parts.igem.org/Part:BBa_K1424005">BBa_K1424005</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 
 +
</div>
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</div>
 +
 
 +
 
 +
<div id="week24entry" class="nb-week" style="display: block">
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<div class="twelve columns">
 +
<h3 class="centered">DNA concentration</h3>
 +
 
 +
<div class="entry pc-calc">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> After measuring the concentration of a sample of DNA in ng/ul with the NanoDrop method, the concentration in terms of pmolar could be determined with the equation
 +
<img src="https://static.igem.org/mediawiki/2014/c/c5/Eqn3.gif">
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</p>
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</div>
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Revision as of 21:30, 22 June 2015

Protocols

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Lysogeny Broth (LB)

Recipe
Antibiotic additions
Antibiotic Stock concentration Final concentration Dillution factor Solvent Storage temperature
Ampicillin 50 mg / mL 50 μg / mL 1000 Filter sterilized H2O 4 °C
Chloramphenicol30 mg / mL30 μg / mL 1000Ethanol-20 °C
Kanamycin50 mg / mL30 μg / mL1000Filter sterilized H2O4 °C
Spectinomycin50 mg / mL50 μg / mL1000Filter sterilized H2O4 °C

Ingredients:

  • Tryptone (10g)
  • NaCl (10g)
  • Yeast Extract (5g)

  1. Fill with 1 L of distilled / filtered H2O.
  2. Autoclave at 121 °C for 20 minutes.
  3. Add antibiotics if needed, after the medium has cooled down.

SOC medium

Recipe
show technical details
{{{tech}}}

Ingredients (1L):

  • Bactotryptone (20 g)
  • Yeast extract (5g)
  • NaCl (0.584g)
  • KCl (0.186g)
  • Agar (20g ONLY IF MAKING PLATES)

  1. Fill with 0.5 L of distilled / filtered H2O.
  2. Autoclave at 121 °C for 20 minutes.
  3. Add 10 mL 1M MgCl2, 10 mL MgSO4 and 20 mL 1M glucose (all should be sterile) prior to use

yB medium

Recipe
{{{tech}}}

Ingredients:

  • Yeast extract (2.5g)
  • Bactotryptone (10g)
  • KCL (0.38g)

  1. Fill with 0.5 L of distilled / filtered H2O.
  2. Add KOH until the pH is 7.4, then autoclave at 121 °C for 20 minutes.
  3. Add 17 mL sterile 1M MgSO4

Synechocystis medium

Recipe
{{{tech}}}

To grow Synechocystis cultures, BG-11 medium was made according to the recipe found on the PhotoSynLab wiki.

Gibson Assembly

{{{tech}}}

The Gibson assembly protocol can be found at New England Biolabs.

DNA isolation and cleaning

{{{tech}}}

For plasmid isolation, the Promega Wizard Plus SV Minipreps DNA Purification System A1460 Miniprep protocol was used. For PCR product isolation, the QIAquick PCR Purification kit was used.

DNA digestion

{{{tech}}}

DNA digests for both ligation and verification used the protocol in the PhotoSynLab wiki.

PCR

{{{tech}}}

The touchdown PCR procedure detailed on the PhotoSynLab wiki was used to amplify DNA.

Nanodrop

{{{tech}}}

A NanoDrop ND-1000 Spectrophotometer was used to determine DNA concentrations.

3A assembly

{{{tech}}}

3A assembly was conducted according to the iGEM 3A assembly protocol.

Ligation

{{{tech}}}

Ligation was performed according to the protocol outlined on the PhotoSynLab wiki.

Transformation (Escherichia coli)

{{{tech}}}

The protocols used for preparing competent DH5a cells, as well as the heat-shock transformation procedure employed can be found at the PhotoSynLab wiki.

Transformation (Synechocystis sp. PCC 6803)

{{{tech}}}

The transformation procedure for transforming Synechocystis can be found at the PhotoSynLab wiki.

Right flank

{{{tech}}}

BBa_K1424001.

Left flank

{{{tech}}}

BBa_K1424000.

Kanamycin resistance

{{{tech}}}

BBa_K1424003.

Lac promotor

{{{tech}}}

BBa_K1424002.

Glucose oxidase

{{{tech}}}

BBa_K1424004.

Left flank + Kanamycin resistance + Right flank

{{{tech}}}

BBa_K1424005.

DNA concentration

{{{tech}}}

After measuring the concentration of a sample of DNA in ng/ul with the NanoDrop method, the concentration in terms of pmolar could be determined with the equation