Team:NTNU Trondheim/Protocols

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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project/Modelling">Modelling</a>
<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project/Modelling">Modelling</a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project/Outreach">Outreach</a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project/Considerations">Considerations</a>
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</li>
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<li class="pg-project_future">
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project/Future">Future Applications</a>
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<li class="pg-project_safety">
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<li class="has-flyout pg-outreach pg-outreach_presentations pg-outreach_panels pg-outreach_awareness">
<li class="has-flyout pg-outreach pg-outreach_presentations pg-outreach_panels pg-outreach_awareness">
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Sponsors">Acknowledgements</a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Acknowledgements/Sponsors">Acknowledgements</a>
<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project" class="flyout-toggle"><span> </span></a>
<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project" class="flyout-toggle"><span> </span></a>
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</li>
</li>
<li id="week1" onclick="weekFilter(this)">
<li id="week1" onclick="weekFilter(this)">
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<a class="nb-week" href="#1">Liquid Broth (LB)</a>
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<a class="nb-week" href="#1">Lysogeny Broth (LB)</a>
</li>
</li>
<li id="week2" onclick="weekFilter(this)">
<li id="week2" onclick="weekFilter(this)">
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<a class="nb-week" href="#2">Super Optimal Broth (S.O.B.)</a>
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<a class="nb-week" href="#2">SOC medium</a>
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<li id="week3" onclick="weekFilter(this)">
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<a class="nb-week" href="#14">Transformation (<i>Synechocystis sp. PCC 6803</i>)</a>
<a class="nb-week" href="#14">Transformation (<i>Synechocystis sp. PCC 6803</i>)</a>
</li>
</li>
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<li id="week15" onclick="weekFilter(this)">
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<a class="nb-week" href="#15">Gel electrophoresis</a>
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<li class="nb-month">
<li class="nb-month">
<div style="top:52px;margin-left:-9px">Plasmids</div>
<div style="top:52px;margin-left:-9px">Plasmids</div>
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</li>
</li>
<li id="week18" onclick="weekFilter(this)">
<li id="week18" onclick="weekFilter(this)">
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<a class="nb-week" href="#18">Kanamycin</a>
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<a class="nb-week" href="#18">Kanamycin resistance</a>
</li>
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<li id="week19" onclick="weekFilter(this)">
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<a class="nb-week" href="#19">Lac inducible promoter</a>
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<a class="nb-week" href="#19">Lac promoter</a>
</li>
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<li id="week20" onclick="weekFilter(this)">
<li id="week20" onclick="weekFilter(this)">
<a class="nb-week" href="#20">Glucose oxidase</a>
<a class="nb-week" href="#20">Glucose oxidase</a>
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<li id="week21" onclick="weekFilter(this)">
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<div style="top:52px;margin-left:-22px">Organisms</div>
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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 id="week21" onclick="weekFilter(this)">
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<a class="nb-week" href="#21"><i>Escherichia coli</i> DH5&#945;</a>
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<li id="week22" onclick="weekFilter(this)">
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<a class="nb-week" href="#22"><i>Synechocystis sp. PCC 6803</i></a>
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</li>
</li>
<li class="nb-month">
<li class="nb-month">
 +
<div style="top:55px;margin-left:-29px">Calculations</div>
<div style="top:55px;margin-left:-29px">Calculations</div>
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<li id="week23" onclick="weekFilter(this)">
 
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<a class="nb-week" href="#23">Transformation efficiency</a>
 
</li>
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<li id="week24" onclick="weekFilter(this)">
<li id="week24" onclick="weekFilter(this)">
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<a class="nb-week" href="#24">Enzyme amount</a>
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<a class="nb-week" href="#24">DNA concentration</a>
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<li id="week25" onclick="weekFilter(this)">
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<a class="nb-week" href="#25">DNA concentration</a>
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<div>Techniques</div>
<div>Techniques</div>
<div>Plasmids</div>
<div>Plasmids</div>
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<div>Organisms</div>
 
<div>Calculations</div>
<div>Calculations</div>
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<div id="week1entry" class="nb-week" style="display: block">
<div id="week1entry" class="nb-week" style="display: block">
<div class="twelve columns">
<div class="twelve columns">
-
<h3 class="centered">Liquid Broth (LB)</h3>
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<h3 class="centered">Lysogeny Broth (LB)</h3>
                                          
                                          
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<div id="week2entry" class="nb-week" style="display: block">
<div class="twelve columns">
<div class="twelve columns">
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<h3 class="centered">Super Optimal Broth (S.O.B.)</h3>
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<h3 class="centered">SOC medium</h3>
</p>
</p>
<div class="entry pc-media">
<div class="entry pc-media">
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</h6>
</h6>
<div class="nb-tech">{{{tech}}}</div>
<div class="nb-tech">{{{tech}}}</div>
-
<p> Made LB plates with ampicillin and ampicillin + kanamycin.
+
<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>
</p>
</div>
</div>
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<div>
<div>
<h6>Recipe
<h6>Recipe
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</h6>
</h6>
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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>   
<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>
</ol>
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</p>
</p>
</p>
</div>
</div>
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</div>
</div>
</div>
 +
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<div id="week4entry" class="nb-week" style="display: block">
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<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>
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</div>
 +
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</div>
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</div>
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<div id="week6entry" class="nb-week" style="display: block">
<div id="week6entry" class="nb-week" style="display: block">
<div class="twelve columns">
<div class="twelve columns">
<h3 class="centered">Gibson Assembly</h3>
<h3 class="centered">Gibson Assembly</h3>
-
</p>
 
<div class="entry pc-techniques">
<div class="entry pc-techniques">
<div>
<div>
-
<h6>Protocol from New England Biolabs
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<h6>
-
<div class="nb-onetech-i nohilite">show technical details</div>
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</h6>
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</h6>
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<div class="nb-tech">{{{tech}}}</div>
<div class="nb-tech">{{{tech}}}</div>
-
<p>
 
-
<ol>
 
-
<br><li>Set up the following reaction on ice:</li> <br>
 
-
</p>
 
<p>
<p>
-
<table width="100%" border="0" cellspacing="0" cellpadding="0" align="center">
+
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>.
-
        <tbody>
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-
            <tr>
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-
                <td rowspan="2">&nbsp;</td>
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-
                <td colspan="3" align="center">Recommended Amount of Fragments Used for Assembly</td>
+
-
            </tr>
+
-
            <tr>
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-
                <td align="center">2-3 Fragment Assembly</td>
+
-
                <td align="center">4-6 Fragment Assembly</td>
+
-
                <td align="center">Positive Control**</td>
+
-
            </tr>
+
-
            <tr>
+
-
                <td>Total Amount of Fragments</td>
+
-
                <td align="right">0.02–0.5 pmols*<br>
+
-
                X μl</td>
+
-
                <td align="right">0.2–1 pmols*<br>
+
-
                X μl</td>
+
-
                <td align="center">10 μl</td>
+
-
            </tr>
+
-
            <tr>
+
-
                <td>Gibson Assembly Master Mix (2X)</td>
+
-
                <td align="right">10 μl</td>
+
-
                <td align="right">10 μl</td>
+
-
                <td align="center">10 μl</td>
+
-
            </tr>
+
-
            <tr>
+
-
                <td>Deionized H<sub>2</sub>O</td>
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-
                <td align="right">10-X μl</td>
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                <td align="right">10-X μl</td>
+
-
                <td align="center">0</td>
+
-
            </tr>
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-
            <tr>
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-
                <td>Total Volume</td>
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-
                <td align="right">20 μL***</td>
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-
                <td align="right">20 μL***</td>
+
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                <td align="center">20 μL</td>
+
-
            </tr>
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        </tbody>
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    </table>
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-
    * Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.<br>
+
-
** Control reagents are provided for 5 experiments.<br>
+
-
*** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
+
</p>
</p>
-
<br>
 
-
<li>Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation. <br><br>
 
-
<i>Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see <a href="https://www.neb.com/tools-and-resources/search?type=FAQ">FAQ</a> section).</i>
 
-
</li><br>
 
-
<li>
 
-
Transform NEB 5-alpha Competent <i>E. coli</i> cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol.
 
-
</li>
 
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</ol>
 
</div>
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<h3 class="centered">DNA isolation and cleaning</h3>
<h3 class="centered">DNA isolation and cleaning</h3>
</p>
</p>
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<div class="entry pc-media">
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<div class="entry pc-techniques">
<div>
<div>
-
<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>
<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> 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 id="week8entry" class="nb-week" style="display: block">
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<div class="twelve columns">
 +
<h3 class="centered">DNA digestion</h3>
 +
</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>
 +
 +
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>
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</div>
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</div>
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</div>
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<div id="week9entry" class="nb-week" style="display: block">
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<div class="twelve columns">
 +
<h3 class="centered">PCR</h3>
 +
</p>
 +
<div class="entry pc-techniques">
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<div>
 +
 +
<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.
 +
</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">
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<div class="twelve columns">
 +
<h3 class="centered">Nanodrop</h3>
 +
</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.
 +
</p>
 +
</div>
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</div>
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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>
 +
</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>
 +
<p> 3A assembly was conducted according to the  <a href="http://parts.igem.org/Help:Protocol/3A_Assembly">iGEM 3A assembly protocol</a>.
 +
</p>
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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">
 +
<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>.
 +
</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">
 +
<h3 class="centered">Transformation (Escherichia coli)</h3>
 +
</p>
 +
<div class="entry pc-techniques">
 +
<div>
 +
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<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>
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</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>
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</div>
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</div>
 +
 +
<div id="week16entry" class="nb-week" style="display: block">
 +
<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>
 +
</div>
 +
</div>
 +
 +
</div>
 +
</div>
 +
 +
<div id="week20entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<h3 class="centered">Glucose oxidase</h3>
 +
 +
<div class="entry pc-plas">
 +
<div>
 +
 +
<div class="nb-tech">{{{tech}}}</div>
 +
<p> <a href="http://parts.igem.org/Part:BBa_K1424004">BBa_K1424004</a>.
 +
</p>
 +
</div>
 +
</div>
 +
 +
</div>
 +
</div>
 +
 +
<div id="week21entry" class="nb-week" style="display: block">
 +
<div class="twelve columns">
 +
<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>
 +
</div>
 +
 +
 +
<div id="week24entry" class="nb-week" style="display: block">
 +
<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">
</p>
</p>
</div>
</div>

Latest revision as of 21:39, 17 October 2014

Team:NTNU Trondheim/Protocols - 2014.igem.org

 

Team:NTNU Trondheim/Protocols

From 2014.igem.org

Team:NTNU_Trondheim/Protocols - 2014.igem.org

 

Team:NTNU_Trondheim/Protocols

From 2014.igem.org

NTNU Genetically Engineered Machines

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

Retrieved from "https://2014.igem.org/Team:NTNU_Trondheim/Protocols"

Retrieved from "https://2014.igem.org/Team:NTNU_Trondheim/Protocols"

Retrieved from "http://2014.igem.org/Team:NTNU_Trondheim/Protocols"