Team:York/Cake

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                                                        <li><a href="https://2014.igem.org/Team:York/Project">Background</a></li>
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                                                        <li><a href="https://2014.igem.org/Team:York/Constructs">Constructs</a></li>
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                                                        <li><a href="https://2014.igem.org/Team:York/Application">Practical Application</a></li>
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<li> <a href="https://2014.igem.org/Team:York/Sustainability">Sustainability</a></li>
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                                        <li><a href="https://twitter.com/iGEMyork" target="_blank"><i class="fa fa-twitter fa-lg"></i></a></li>
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                                        <li><a href="mailto:igemyork2014@gmail.co.uk" target="_blank"><i class="fa fa-envelope fa-lg"></i></a></li>
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<div class="col-lg-12">
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<!-- Put all the content under here... -->
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<ul class="cb-slideshow">
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<h1> Laboratory Protocols </h1>
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            <li><span>Image 01</span></li>
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<ul class="nav nav-tabs" role="tablist" id="myTab">
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            <li><span>Image 02</span></li>
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    <li class="active"><a href="#one" role="tab" data-toggle="tab">LB Media</a></li>
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            <li><span>Image 03</span></li>
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    <li><a href="#two" role="tab" data-toggle="tab">LA Media</a></li>
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            <li><span>Image 04</span></li>
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    <li><a href="#three" role="tab" data-toggle="tab">Plasmid Purification</a></li>
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            <li><span>Image 05</span></li>
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    <li><a href="#gel" role="tab" data-toggle="tab">Gel Electrophoresis</a></li>
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            <li><span>Image 06</span></li>
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    <li><a href="#four" role="tab" data-toggle="tab">Gel Extraction</a></li>
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            <li><span>Image 07</span></li>
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    <li><a href="#five" role="tab" data-toggle="tab">SOC Media</a></li>
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            <li><span>Image 08</span></li>
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    <li><a href="#six" role="tab" data-toggle="tab">Competent Cells</a></li>
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    <div class="container">
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     <div class="tab-pane active" id="one">
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     <div class="jumbotron text-center home">
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        <div class="inner">
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<h2>Lysogeny Broth</h2>
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            <div class="logo-round">
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<h3>Materials</h3>
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                <img src="https://static.igem.org/mediawiki/2014/6/6a/UoYiGEMlogo-noback.png" class="img-responsive center">
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<ul>
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            </div>
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<li>10g of tryptone</li>
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            <h1>Hello! We're from the</h1>
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<li>5g of yeast extract</li>
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                        <a href="http://www.york.ac.uk/biology/" target="_blank"><img src="https://static.igem.org/mediawiki/2014/5/5e/Uoylogo-white.png" class="img-responsive center" style="max-width:600px; margin-top:30px; margin-bottom:30px; margin-left:auto; margin-right:auto;"></a>
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<li>10g of NaCl</li>
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            <hr>
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<li>1L of Deionised Water</li>
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            <p>Our project EcoCADMUS (E. Coli CAdmium DecontaMination Universal System) targets industrial and mineral processing waste-water contamination by heavy metals and sulfates. EcoCADMUS provides a safe and selective way to remove Cadmium Sulfide from waste water using Synthetic Biology.
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</ul>
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<br><br>
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<a href="https://twitter.com/iGEMyork" target="_blank"><i class="fa fa-twitter fa-lg"></i></a> &nbsp; &nbsp; &nbsp; <a href="mailto:igemyork2014@gmail.co.uk" target="_blank"><i class="fa fa-envelope fa-lg"></i></a>
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<h3>Procedure</h3>
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</p>
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<ol>
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                <a href="https://2014.igem.org/Team:York/Project">
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<li>Use a container a container with at least double the volume of the LB that you are making.</li>
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                    <button type="button" class="btn btn-primary">Learn More!</button>
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<li>Measure out the weights of tryptone, yeast extract and sodium chloride as above then fill up with deionised water to 1l and mix well until clear.</li>
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                </a>
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<li>Ensure the lid is unscrewed by two and a half turns</li>
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        </div>
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<li>Send to be autoclaved</li>
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</ol>
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<div class="row home-row">
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<button class="col-md-4 btn btn-primary home-panel">
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<a href="https://2014.igem.org/Team:York/Team">
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<h3>Our Team</h3>
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<img class="img-responsive" src="https://static.igem.org/mediawiki/2014/4/44/York_TeamHomePage.jpg">
 +
<p>Come and meet our lovely team!<br>We've been working hard (mostly!) on our project all over Summer.</p>
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</a>
 +
</button>
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<button class="col-md-4 btn btn-primary home-panel">
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<a href="https://2014.igem.org/Team:York/Constructs">
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<h3>Plasmid Construct</h3>
 +
<img class="img-responsive" src="https://static.igem.org/mediawiki/2014/a/ae/York_Constructs.jpg">
 +
<p>Look at the plasmid we're using in our project, complete with a repertoire of Cadmium related genes.</p>
 +
</a>
 +
</button>
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<button class="col-md-4 btn btn-primary home-panel">
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<a href="https://2014.igem.org/Team:York/SocialImpacts">
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<h3>Social Outreach</h3>
 +
<img class="img-responsive" src="https://static.igem.org/mediawiki/2014/7/7d/York_Researchhomepage.jpg">
 +
<p>We've been busy communicating Synthetic Biology and contemplating the sustainability and ethics of our project. </p>
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</a>
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</button>
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    <div class="tab-pane" id="two">
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        <div class="filter"></div>
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</body>
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<h2>Lysogeny Agar</h2>
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<h3>Materials</h3>
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<ul>
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<li>10g of tryptone</li>
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<li>5g of yeast extract</li>
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<li>10g of NaCl</li>
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<li>15g of agar </li>
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<li>1L of Deionised Water</li>
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</ul>
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<h3>Procedure</h3>
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<ol>
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<li>Use a container a container with at least double the volume of the LA that you are making.</li>
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<li>Measure out the weights of tryptone, yeast extract, sodium chloride and agar with deionised water to 1l and mix well.</li>
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<li>Ensure the lid is unscrewed by two and a half turns.</li>
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<li>Send to be autoclaved.</li>
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<li>Pour the plates next to a Bunsen burner. </li>
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<li>Leave for 15-20 minutes to set/solidify. </li>
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</ol>
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    <div class="tab-pane" id="three">
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<h2>Mini-Prep or Plasmid Purification</h2>
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<ul>
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<li>Harvest bacterial cells<br>
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1. Pellet 20ml of saturated E. coli for 60 seconds  at 11,000 x g.<br>
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2. Discard supernatant and remove as much liquid as possible.</li>
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<li>Lyse cells<br>
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1. Add 500ml Resuspension Buffer P1 and resuspend cell pellet by vortexing.<br>
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2. Split the solution into two 1.5ml microcentrifuge tubes.<br>
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3. Add 250μl Lysis Buffer 2. <br>
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4. Mix gently by inverting tube 8 times. <br>
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5. Incubate at room temperature for five minutes or until lysate appears clear.<br>
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6. Add 300μl Neutralization Buffer 3.<br>
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7. Mix thoroughly by inverting tube 8 times.</li>
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<li>Clarification of lysate<br>
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1. Centrifuge for five minutes at 11,000 x g at room temperature<br>
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2. Put 500μl of Buffer PW1 per 1.5ml microcentrifuge tube used in heat block heated to 50օC</li>
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<li>Bind DNA<br>
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1. Place ISOLATE II Plasmid Mini Spin Column in a 2ml Collection Tube<br>
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2. Pipette a maximum of 750μl of clarified sample supernatant onto column<br>
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3. Incubate at room temperature for two minutes.<br>
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4. Centrifuge for one minute at 11,000 x g and discard flow-through.<br>
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5. Repeat stage 4 using the same ISOLATE II Plasmid Mini Spin Column and 2ml Collection Tube with the clarified sample supernatant from the other 1.5ml microcentrifuge tube from the same sample.</li>
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<li>Wash silica membrane<br>
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1. Add 500μl Wash Buffer Pw1<br>
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2. Centrifuge for one minute at 11,000 x g <br>
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3. Add 600μl Wash Buffer PW2 (supplemented with ethanol)<br>
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4. Centrifuge for one minute at 11,000 x g <br>
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5. Discard flow-through and reuse Collection Tube</li>
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<li>Dry silica membrane<br>
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1. Centrifuge for two minutes at 11,000 x g, to remove residual ethanol<br>
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-
2. Place ISOLATE II Plasmid Mini Spin Column in a 1.5ml microcentrifuge tube.</li>
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<li>Elute DNA<br>
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1. Add 50μl Elution Buffer P directly on the top of the silicon matrix<br>
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2. Incubate at room temperature for two minutes<br>
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3. Centrifuge for one minute at 11,000 x g.</li>
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</ul>
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    <div class="tab-pane" id="gel">
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<h2>Gel Electrophoresis</h2>
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<h3>Materials</h3>
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For a 1% Agarose Gel:
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<ul>
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<li>1g Agarose</li>
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<li>100ml de-ionised water</li>
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<li>10&#956;l sybrsafe&#8482;</li>
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<li>Loading Buffer</li>
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<li>Masking Tape</li>
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</ul>
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<h3>Procedure</h3>
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<b>Make 1% Agarose Gel:</b>
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<ol>
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<li>Dissolve 1g of agarose in 100ml of deionised water.</li>
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<li>Microwave for 2 minutes and check it is all dissolved.</li>
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<li>Wait for it to cool</li>
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<li>Add the sybrsafe (10&#8482;l) pour the gel into the mold and leave it to set for 15 minutes.</li>
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</ol>
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<b>Preparing DNA samples to load into wells in the gel.</b><br>
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Add loading buffer to your DNA samples to help visualise the DNA running through the gel.<br>
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<b>Performing gel electrophoresis:</b>
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<ol>
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<li>Inject your DNA samples into the appropriate wells and use a HyperLadder for reference (left hand side).</li>
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<li>Turn on the machine and make sure the black lead is attached to the black end and the red lead is attached to the red end. <i>Black is negative, Red is positive.</i> The DNA will move towards the red because it is negative.</li>
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<li>Leave gel running for at around 30 minutes.</li>
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<li>Take to U:Genius Image Capture in biolab one to see the DNA bands under UV light. Do not leave the UV light on for too long before taking the photo as this can degrade the DNA.</li>
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</ol>
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    </div>
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    <div class="tab-pane" id="four">
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<h2>Agarose Gel Extraction</h2>
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1. Excise and dissolve gel slice<br>
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2. Using a clean scalpel excise DNA fragment from gel<br>
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3. Remove excess agarose, determine weight of gel slice and transfer into a clean tube<br>
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4. Add 200μl Binding Buffer CB per 100mg of 2% agarose gel<br>
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5. Incubate sample at 50օC for ten minutes, vortexing sample briefly every three minutes until gel slice is completely dissolved<br>
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6. Incubate at room temperature for two minutes<br>
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<strong>Bind DNA</strong><br>
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1. Place ISOLATE II PCR and Gel Column in a 2ml Collection Tube and load 600μl of the sample<br>
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2. Centrifuge for thirty seconds at 11,000 x g and discard flow-through<br>
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-
3. Reuse collection tube for step 3<br>
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-
<strong>Wash silica membrane</strong><br>
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-
1. Add 700μl Wash Buffer CW to ISOLATE II PCR and Gel Column<br>
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-
2. Centrifuge for thirty seconds at 11,000 x g<br>
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-
3. Discard flow-through and place column back into collection tube<br>
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4. Repeat step three to minimize chaotropic salt carry-over<br>
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<strong> Dry silica membrane</strong><br>
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-
1. Centrifuge for one minute at 11,000 x g, to remove residual ethanol<br>
+
-
2. Place ISOLATE II PCR and Gel Column in a 1.5ml microcentrifuge tube<br>
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<h3>Elute DNA</h3>
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-
1. Incubate at room temperature for three minutes <br>
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-
2. Add 15-30μl Elution Buffer C directly onto silica membrane<br>
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-
3. Incubate at room temperature for three minutes<br>
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4. Centrifuge for one minute at 11,000 x g.
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    <div class="tab-pane" id="five">
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<h2>SOC Media</h2>
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<h3>Materials</h3>
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<p>To make 100ml SOC Media</p>
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<ul>
+
-
<li>2g Tryptone</li>
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<li>0.5g Yeast Extract</li>
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<li>2.5ml 400mM NaCl</li>
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<li>625&#956;l 400mM KCl</li>
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<li>10ml 100mM MgCl<small>2</small></li>
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<li>1ml 200mM Autoclaved and filter sterilised Glucose</li>
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-
</ul>
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-
 
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-
<h3>Procedure</h3>
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-
<ol>
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-
<li>Weigh out tryptone and yeast extract into vessel suitable for autoclaving. Add the NaCl, KCl, MgCl<small>2</small> to the bottle.</li>
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-
<li>Make up to 100ml with Distilled Water.</li>
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<li>Make glucose solution in a vessel suitable for autoclaving.</li>
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-
<li>Autoclave both solutions separately to avoid the reaction of glucose with other components.</li>
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<li>Add 1ml glucose solution using a filter sterilisation syringe to the media.</li>
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</ol>
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    </div>
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    <div class="tab-pane" id="six">
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<h2>Competent Cell Production</h2>
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<h3>Materials</h3>
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<ul>
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-
<li>100ml LB + 5ml for overnight culture</li>
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<li>100mM CaCl<small>2</small></li>
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<li>85mM CaCl<small>2</small>, 15% glycerol v/v
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</ul>
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-
<h3>Procedure</h3>
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-
<ol>
+
-
<li>Streak competent cells onto agar plate and incubate overnight at 37 <small>O</small>C</li>
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-
<li>Prepare and autoclave above solutions.<br>
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-
Inoculate a single colony into 5ml LB in a 50 ml falcon tube. Grow overnight at 37 <small>O</small>C, shaking at 200rpm.<br>
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-
Keep solutions at 4 <small>O</small>C overnight, and LB at 37 <small>O</small>C so that when cells get transferred they do not experience a temperature change.</li>
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-
<li>Pre-cool the rotor of the centrifuge.<br>
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-
Use 1 ml of overnight culture to inoculate 100ml of LB in a 250ml bottle. Shake at 37 <small>O</small>C for 1.5-3 hours, until OD650 reaches 0.4-0.6.<br>
+
-
Put cells on ice for 10 mins (keep cold from now on, and cool everything on ice before adding). Split into 2 x 50ml falcon tubes.<br>
+
-
Centrifuge in the big centrifuge for 3 mins at 5000 rpm.<br>
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-
Decant supernatant and gently resuspend in 5ml cold 100mM CaCl<small>2</small> by inverting tube slowly. (Cells susceptible to mechanical disruption)<br>
+
-
Incubate on ice for 20mins<br>
+
-
Centrifuge as before (3 mins at 5000 rpm)<br>
+
-
Discard supernatant and resuspend in 2.5ml cold 100mM CaCl<small>2</small>/ 15% glycerol v/v<br>
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-
Pipette into microtubes and freeze in -80<small>O</small>C. (100µl per tube).</li>
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</ol>
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Latest revision as of 13:20, 17 October 2014

Team York 2014

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Hello! We're from the

Our project EcoCADMUS (E. Coli CAdmium DecontaMination Universal System) targets industrial and mineral processing waste-water contamination by heavy metals and sulfates. EcoCADMUS provides a safe and selective way to remove Cadmium Sulfide from waste water using Synthetic Biology.

     

Retrieved from "http://2014.igem.org/Team:York/Cake"