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

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{{NTNU Trondheim_Draft}}
 
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<div style="top:67px;margin-left:-6px">Spring</div>
 
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<a class="nb-week" href="#1">Lysogeny Broth (LB)</a>
 
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<a class="nb-week" href="#2">SOC medium</a>
 
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<a class="nb-week" href="#3">yB medium</a>
 
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<a class="nb-week" href="#4"><i>Synechocystis</i> medium</a>
 
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<div style="top:140px;margin-left:-18px">Techniques</div>
 
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<a class="nb-week" href="#6">Gibson assembly</a>
 
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<a class="nb-week" href="#7">DNA isolation and cleaning</a>
 
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<a class="nb-week" href="#8">DNA digestion</a>
 
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<a class="nb-week" href="#9">PCR</a>
 
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<a class="nb-week" href="#10">NanoDrop</a>
 
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<a class="nb-week" href="#11">3A assembly</a>
 
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<a class="nb-week" href="#12">Ligation</a>
 
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<a class="nb-week" href="#13">Transformation (<i>Escherichia coli</i>)</a>
 
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<a class="nb-week" href="#14">Transformation (<i>Synechocystis sp. PCC 6803</i>)</a>
 
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<div style="top:52px;margin-left:-9px">Plasmids</div>
 
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<a class="nb-week" href="#16">Right flank</a>
 
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<a class="nb-week" href="#17">Left flank</a>
 
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<a class="nb-week" href="#18">Kanamycin resistance</a>
 
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<a class="nb-week" href="#19">Lac promoter</a>
 
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<a class="nb-week" href="#20">Glucose oxidase</a>
 
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<a class="nb-week" href="#21">Left flank + Kan + Right flank</a>
 
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<div style="top:55px;margin-left:-29px">Calculations</div>
 
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<a class="nb-week" href="#24">DNA concentration</a>
 
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<div class="ten columns team-bios-container">
 
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<h2 class="centered">Protocols</h2>
 
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<h5 style="margin-left:15px;text-align:left">Filter by category:
 
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<div>Media</div>
 
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<div>Techniques</div>
 
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<img id="plas" src="https://static.igem.org/mediawiki/2015/7/74/NTNU_Trondheim_plasmids_on.png">
 
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<div id="week1entry" class="nb-week" style="display: block">
 
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<h3 class="centered">Lysogeny Broth (LB)</h3>
 
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<h6>Recipe
 
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<div class="nb-onetech-i nohilite">Antibiotic additions</div>
 
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<div class="AB-table">
 
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<th> Antibiotic</th> <th> Stock concentration</th> <th>Final concentration</th> <th>Dillution factor</th> <th>Solvent</th> <th>Storage temperature</th>
 
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<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>
 
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</tr>
 
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<tr>
 
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<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>
 
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</tr>
 
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<tr>
 
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<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>
 
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</tr>
 
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<tr>
 
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<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>
 
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<p>
 
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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>
 
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</ol></p>
 
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<h3 class="centered">SOC medium</h3>
 
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</p>
 
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<div class="entry pc-media">
 
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<h6>Recipe
 
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<div class="nb-onetech-i nohilite">show technical details</div>
 
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</h6>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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> 
 
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<div id="week3entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">yB medium</h3>
 
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</p>
 
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<div class="entry pc-media">
 
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<div>
 
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<h6>Recipe
 
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</h6>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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> 
 
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</ol>
 
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</div>
 
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<div id="week4entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Synechocystis medium</h3>
 
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</p>
 
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<div class="entry pc-media">
 
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<div>
 
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<h6>Recipe
 
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</h6>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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>.
 
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</p>
 
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</div>
 
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<div id="week6entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Gibson Assembly</h3>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<h6>
 
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</h6>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<p>
 
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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>.
 
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</p>
 
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<div id="week7entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">DNA isolation and cleaning</h3>
 
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</p>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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.
 
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</p>
 
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</div>
 
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</div>
 
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</div>
 
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<div id="week8entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">DNA digestion</h3>
 
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</p>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<p>
 
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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>.
 
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<div id="week9entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">PCR</h3>
 
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</p>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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.
 
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</p>
 
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<div id="week10entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Nanodrop</h3>
 
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</p>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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.
 
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</p>
 
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</div>
 
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</div>
 
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<div id="week11entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">3A assembly</h3>
 
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</p>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<p> 3A assembly was conducted according to the  <a href="http://parts.igem.org/Help:Protocol/3A_Assembly">iGEM 3A assembly protocol</a>.
 
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</p>
 
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</div>
 
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</div>
 
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</div>
 
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</div>
 
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<div id="week12entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Ligation</h3>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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>.
 
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</p>
 
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</div>
 
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</div>
 
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</div>
 
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</div>
 
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<div id="week13entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Transformation (Escherichia coli)</h3>
 
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</p>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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>.
 
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</p>
 
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</div>
 
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</div>
 
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</div>
 
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<div id="week14entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Transformation (Synechocystis sp. PCC 6803)</h3>
 
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</p>
 
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<div class="entry pc-techniques">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<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>.
 
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</p>
 
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</div>
 
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<div id="week16entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Right flank</h3>
 
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<div class="entry pc-plas">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<p> <a href="http://parts.igem.org/Part:BBa_K1424001">BBa_K1424001</a>.
 
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</p>
 
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<div id="week17entry" class="nb-week" style="display: block">
 
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<h3 class="centered">Left flank</h3>
 
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<div class="entry pc-plas">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<p> <a href="http://parts.igem.org/Part:BBa_K1424000">BBa_K1424000</a>.
 
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</p>
 
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</div>
 
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</div>
 
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<div id="week18entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Kanamycin resistance</h3>
 
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<div class="entry pc-plas">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<p> <a href="http://parts.igem.org/Part:BBa_K1424003">BBa_K1424003</a>.
 
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</p>
 
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</div>
 
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</div>
 
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<div id="week19entry" class="nb-week" style="display: block">
 
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<div class="twelve columns">
 
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<h3 class="centered">Lac promotor</h3>
 
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<div class="entry pc-plas">
 
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<div>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<p> <a href="http://parts.igem.org/Part:BBa_K1424002">BBa_K1424002</a>.
 
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</p>
 
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<div id="week20entry" class="nb-week" style="display: block">
 
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<p> <a href="http://parts.igem.org/Part:BBa_K1424005">BBa_K1424005</a>.
 
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<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
 
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Retrieved from "<a href="https://2013.igem.org/Team:Cornell/notebook">https://2013.igem.org/Team:Cornell/notebook</a>" and "<a href="https://2014.igem.org/Team:NTNU_Trondheim/notebook">https://2014.igem.org/Team:NTNU_Trondheim/notebook</a>"</div>
 
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Latest revision as of 19:03, 16 September 2015