Difference between revisions of "Team:Warwick/Experiments"

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{{Warwick}}
 
 
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<link rel="stylesheet" href="https://2018.igem.org/Team:Warwick/CSS?action=raw&ctype=text/css">
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<div class="column full_size">
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<script>
  
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<h1>Experiments</h1>
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function goToNotebook(){
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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}
  
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<p>
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function goToResults(){
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Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.  
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}
  
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<h3>What should this page contain?</h3>
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<ul>
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<li> Protocols </li>
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<li> Experiments </li>
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<li> Documentation of the development of your project </li>
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<h3>Inspiration</h3>
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<ul>
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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break;
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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case 1:
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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        window.location = 'https://2018.igem.org/Team:Warwick/Description';
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        window.location = 'https://2018.igem.org/Team:Warwick/Design';
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        window.location = 'https://2018.igem.org/Team:Warwick/Experiments';
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        window.location = 'https://2018.igem.org/Team:Warwick/InterLab';
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        window.location = 'https://2018.igem.org/Team:Warwick/Model';
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        window.location = "https://2018.igem.org/Team:Warwick";
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        window.location = "https://2018.igem.org/Team:Warwick/Medals";
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        $("#Content1").html("<h2>PCR protocol</h2><p>For the most part, out PCRs were performed using Q5 polymerase from NEB. Master mix was created according to manufacturer specifications this was added to samples of DNA. Samples were incubated in a thermocycler with programs set up according to manufacturer instructions and extension times and annealing temperatures were adjusted based on the properties of the DNA being amplified.</p>");
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        $("#Content2").html("<h2>Gel Electrophoresis</h2><p>Gel was prepared by adding 0.5g of agarose powder to 50ml of TAE buffer. The mixture was heated until the powder had dissolved. 5ul of SYBR SAFE gel stain from NEB was added and the gel was left to set in a gel tray. The gel was placed in an electrophoresis bath filled with TAE buffer and a constant voltage of 120V was applied for forty minutes. The gel was then analysed using a gel imaging machine to determine results.</p>");
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        $("#Content3").html("<h2>Plasmid miniprep</h2><p>Plasmids were harvested from cultured cells using both Qiagen and NEB miniprep kits, according to the manufacturer’s instructions.</p>");
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        $("#Content4").html("<h2>PCR Purification</h2><p>PCR products were purified using NEB PCR cleanup kits, according to the manufacturer’s instructions.</p>");
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        $("#Content").html("<h2>DNA Gel Extraction</h2><p>DNA was retrieved from agarose gels using Qiagen and NEB gel extraction kits, according to the manufacturer’s instructions.</p>");
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        $("#Content").html("<h2>Nanodrop DNA Concentration Testing</h2><p>A blank measurement was made by placing 1ul of NEB elution buffer on the NanoDrop stage and instructing it to make a blank measurement. Then 1ul of DNA in solution was placed on the stage and the NanoDrop machine was instructed to measure the DNA concentration.</p>");
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        $("#Content").html("<h2>Plasmid Assembly</h2><p>Plasmids were assembled using Gibson assembly from NEB. DNA concentration was measured using a NanoDrop machine in order to determine the volume of each solution needed, as per the manufacturer’s recommendations. The master mix was made up to manufacturer’s specifications and the plasmid and DNA was added. The mixture was then incubated at 50°C for fifteen minutes.</p>");
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        $("#Content").html("<h2>Cell Transformation</h2><p>For cell transformation we used both DH5α and BL21 competent cells from NEB. These were transformed using the recommended plasmid concentration and transformation protocol from the manufacturer.</p>");
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        $("#Content").html("<h2>Checking for Oestregen Degradation in Environmental Samples</h2><p>The aim of this experiment was to identify potential bacteria that utilise oestrogen as a carbon source from any samples obtained. This was achieved through creating a minimal medium with any non-carbon based potential nutrients these bacteria may need, aside from a carbon source – which is provided through 17b-estradiol. The process uses serial dilution over time to do this in order to slowly remove the non-degrading bacteria and allow the degrading bacteria to grow.<br>1. Add 1ml of sample to 10ml of MDG medium containing 0.1% 17b-estradiol (0.1g) in sterile Erlenmeyer flask (250ml)<br>2. Add bacteria to shaking incubator and grow at 28°C.<br>3. Allow bacteria to grow until a dense culture is visible (through cloudiness).<br>4. Repeat the first method up to 8 times using 100ul of the bacteria that was allowed to grow until a dense culture was visible, do this until there is no reduction in types of colonies formed.<br>5. After 8 days, form a 101 to 109 serial dilution of samples in physiological saline solution (0.85% NaCl diluted in sterile water).<br>6. Then add 100ul of each dilution to ISP, R2A and YM culture medium and incubate for 5 days.<br>7. Any that have grown are capable of oestrogen degradation.<br><br>Overall this should identify oestrogen degrading bacteria which can then first be sequenced in order to identified through 16s rRNA sequencing.<br><br>This protocol is based on the paper “Degradation of Estrogens by Rhodococcus zopfii and Rhodococcus equi Isolates from Activated Sludge in Wastewater Treatment Plants” by Yoshimoto et al.</p>");
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        $("#Content").html('<h2>References</h2><p>“Degradation of Estrogens by Rhodococcus zopfii and Rhodococcus equi Isolates from Activated Sludge in Wastewater Treatment Plants”, Takeshi Yoshimoto, Fumiko Nagai, Junji Fujimoto, Koichi Watanabe, Harumi Mizukoshi, Takashi Makino, Kazumasa Kimura, Hideyuki Saino, Haruji Sawada, Hiroshi Omura, Appl. Environ. Microbiol. Sep 2004, 70 (9) 5283-5289</p>');
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<h1 id='GenericTitle'>Experiments</h1>
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Revision as of 23:35, 16 October 2018

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Experiments