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− | {{CCU_Taiwan}}
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− | <h1>Experiments</h1> | + | |
− | <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> | + | <script type="text/javascript" src="https://2017.igem.org/Team:CCU_Taiwan/assets/js/js_anchor? |
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| + | <body class="no-sidebar" class="body.homepage"> |
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| + | <div class="fixed" style="cursor:pointer;"> |
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| + | <div id="page-wrapper"> |
| + | |
| + | <!-- Header --> |
| + | <div id="header"> |
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| + | <!-- Inner --> |
| + | <div class="inner"> |
| + | <header> |
| + | <h1><a href="https://2017.igem.org/Team:CCU_Taiwan" id="logo">InterLab</a></h1> |
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| + | </header> |
| + | </div> |
| + | |
| + | <!-- Nav --> |
| + | <nav id="nav"> |
| + | <ul> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan">Home</a></li> |
| + | |
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| + | <li> |
| + | <a href="#">Project</a> |
| + | <ul> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Description">Description</a></li> |
| + | <li> |
| + | <a href="https://2017.igem.org/Team:CCU_Taiwan/Design">Biosensor</a> |
| + | <ul> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Design#CSP">CSP detector</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Design#Lactate">Lactate detector</a></li> |
| + | </ul> |
| + | </li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Test_paper">Test paper</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Experiments">Experiments</a></li> |
| + | |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Results">Results</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Demonstrate">Demonstrate</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Applied_Design">Applied design</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Notebook">Notebook</a></li> |
| + | |
| + | </ul> |
| + | </li> |
| + | |
| + | <li> |
| + | |
| + | <a href="#">Hardware</a> |
| + | <ul> |
| + | |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Hardware">Hardware overview</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Device_design">Device design</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Device_detection">Device detection</a></li> |
| + | </ul> |
| + | </li> |
| + | |
| + | <li> |
| + | <a href="#">Software</a> |
| + | <ul> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/IOT system">IOT system</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/APP">APP</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Machine_learning">Machine learning</a></li> |
| + | </ul> |
| + | </li> |
| + | |
| + | </li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Model">Modeling</a></li> |
| + | |
| + | <li> |
| + | <a href="#">Human practice</a> |
| + | <ul> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Human_Practices">Human practice overview</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/HP/Silver">Silver HP</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/HP/Gold_Integrated">Integrate & Gold</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Engagement">Public engagemant</a></li> |
| + | |
| + | </ul> |
| + | </li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Entrepreneurship">Entrepreneurship</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Safety">Safety</a></li> |
| + | <li> |
| + | <a href="#">Parts</a> |
| + | <ul> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Parts">Parts</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Basic_Part">Basic parts</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Composite_Part">Composite parts</a></li> |
| + | |
| + | </ul> |
| + | </li> |
| + | |
| + | |
| + | |
| + | <li> |
| + | <a href="#">Team</a> |
| + | <ul> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Members">Team members</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Attributions">Attributions</a></li> |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/Collaborations">Collaborations</a></li> |
| + | |
| + | </ul> |
| + | </li> |
| + | |
| + | <li><a href="https://2017.igem.org/Team:CCU_Taiwan/InterLab">InterLab</a></li> |
| + | |
| + | </ul> |
| + | </nav> |
| + | |
| + | </div> |
| + | |
| + | <!-- Main --> |
| + | <div class="wrapper style1"> |
| + | |
| + | <div class="container"> |
| + | <article id="main" class="special"> |
| + | <header> |
| + | |
| + | |
| + | <div class="div_nav"> |
| + | <nav class="toc_nav" id="toc_show"> |
| + | <ul> |
| + | <li> |
| + | <a href="#Fluorescein">Fluorescein curve</a> |
| + | <ul> |
| + | <li><a href="#Fluorescein-Plate-reader">Plate reader</a></li> |
| + | <li><a href="#Fluorescein-Material">Material</a></li> |
| + | <li><a href="#Fluorescein-Method">Method</a></li> |
| + | <li><a href="#Fluorescein-Data-result">Data result</a></li> |
| + | </ul> |
| + | </li> |
| + | <li> |
| + | <a href="#OD600">OD600 Reference point</a> |
| + | <ul> |
| + | <li><a href="#OD600-Plate-reader">Plate reader</a></li> |
| + | <li><a href="#OD600-Material">Material</a></li> |
| + | <li><a href="#OD600-Method">Method</a></li> |
| + | <li><a href="#OD600-Data-result">Data result</a></li> |
| + | </ul> |
| + | </li> |
| + | |
| + | <li> |
| + | <a href="#Cell">Cell measure</a> |
| + | <ul> |
| + | <li><a href="#Cell-Material">Material</a></li> |
| + | <li><a href="#Cell-Method">Method</a></li> |
| + | <li><a href="#Cell-Data-result">Data result</a></li> |
| + | </ul> |
| + | </li> |
| + | |
| + | |
| + | </ul> |
| + | <svg class="toc-marker" width="200" height="200" xmlns="http://www.w3.org/2000/svg"> |
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| + | </svg> |
| + | </nav> |
| + | |
| + | <article class="contents_nav"> |
| + | <section> |
| + | |
| + | <h2>Experiment</h2> |
| + | |
| + | <h3>Transformation ( by 1.5 ml Eppendorf tube)</h3> |
| | | |
| <p> | | <p> |
− | Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.
| + | 1. 2.5 μg DNA + 25 μl competent cell (ECOS 101 DH5α)<br> |
| + | 2. On ice 30 min<br> |
| + | 3. 42℃ heat shock 30 sec<br> |
| + | 4. On ice 5 min<br> |
| + | (In laminar flow)<br> |
| + | 5. Add LB to 500 μl,37℃ 170 rpm incubate 2hour.<br> |
| + | 6. plate 200 μl ( 300 μl remained) spread over plate by spreader.<br> |
| + | 7. Incubate the plate in 37℃ overnight. |
| </p> | | </p> |
| | | |
| + | |
| + | <h3>Patch and small scale culture</h3> |
| + | |
| + | <p> |
| + | 1. Pick a colony by toothpick then “patch” on another agar plate as bacteria stock. Incubate in 37℃ 14 hour~16 hour.<br> |
| + | EX: picture<br> |
| + | 2. Dip the toothpick into 5 ml LB (with antibiotic) as small scale culture for plasmid extraction. Incubate in 37℃ 170rpm for 14 hour~16 hour.<br> |
| + | |
| + | </p> |
| + | |
| + | |
| + | <h3>Mini extraction</h3> |
| + | |
| + | <p> |
| + | Follow the “Biokit” protocol. |
| + | |
| + | </p> |
| + | |
| + | |
| + | <h3>IV. Digestion</h3> |
| + | |
| + | <p> |
| + | 1. 0.5 μg DNA + 0.2 μl fast digestion restriction enzyme ( 10 unit/μl, final 0.25 μg/unit) + 1 μl 10X buffer + mQ H<sub>2</sub>O -> total volume 10 μl. <br> |
| + | 2. Bath in 37℃ warm water for 15 min. |
| + | </p> |
| + | |
| + | |
| + | <h3>Ligation protocol</h3> |
| + | |
| + | <p> |
| + | *The ideal volume for DNA ligation is 10 μl, but it is adding the volume to 15~20 μl is fine, since part of the liquid will vaporize during ligation.<br> |
| + | *It is recommended that the concentration for the backbone is 20 fmol.<br> |
| + | *The molar ratio for backbone : insert = 1 : 3 <br> |
| + | *The ligase required 0.1 ~ 0.5 unit/tube.<br> |
| + | |
| + | 1. Quantify the concentration of the linear DNAs that you are going to ligate.<br> |
| + | 2. Calculate the molar weight of DNA. Converse the unit into ng/fmol.<br> |
| + | 3. Calculate how much Nano gram is required for 20 fmol of backbone and 60 fmol of insert.<br> |
| + | 4. Then by the quantified concentration, find out that how much μl is required.<br> |
| + | 5. Adjust the total volume to 8.9 μl/tube.<br> |
| + | 6. Add 1 μl of 10x ligation buffer and 0.1 μl of T4 ligase (5 unit/μl)<br> |
| + | 7. Place it in 16°C water bath, overnight.<br> |
| + | |
| + | |
| + | </p> |
| + | |
| + | |
| + | |
| + | <h3>Run electrophoresis gel</h3> |
| + | |
| + | <p> |
| + | 1. X (g) agarose + Y (ml) 0.5XTAE make a suitable agarose gel( depend on your DNA size), X/Y = 1.2%~1.5% (<0.5k) heating until all agarose melted.<br> |
| + | 0.8%~1% (0.5~10k)<br> |
| + | 0.3%~0.7% (>10k)<br> |
| + | Cool down the agarose gel (use the back of your hand to check it, as long as it does not burn your hand, it is suitable temperature) + EtBr ( final, 5 μl/100 ml).<br> |
| + | </p> |
| + | |
| + | <p>2. Marker:</p> |
| + | |
| + | <style type="text/css"> |
| + | .tg {border-collapse:collapse;border-spacing:0;border-color:#ccc;} |
| + | .tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#fff;} |
| + | .tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#f0f0f0;} |
| + | .tg .tg-yw4l{vertical-align:top} |
| + | .tg .tg-b7b8{background-color:#f9f9f9;vertical-align:top} |
| + | </style> |
| + | <table class="tg"> |
| + | <tr> |
| + | <th class="tg-yw4l"></th> |
| + | <th class="tg-yw4l">Marker</th> |
| + | <th class="tg-yw4l">Sample</th> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8" rowspan="2"></td> |
| + | <td class="tg-b7b8">Suitable ladder</td> |
| + | <td class="tg-b7b8">DNA</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-yw4l">2 μl</td> |
| + | <td class="tg-yw4l">10 μl</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8">6X dye</td> |
| + | <td class="tg-b7b8">2 μl</td> |
| + | <td class="tg-b7b8">2 μl</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-yw4l">mQ H2O</td> |
| + | <td class="tg-yw4l">8 μl</td> |
| + | <td class="tg-yw4l">0</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8">Total volume</td> |
| + | <td class="tg-b7b8">12 μl</td> |
| + | <td class="tg-b7b8">12 μl</td> |
| + | </tr> |
| + | </table> |
| + | |
| + | <p>3. Load 12 μl per well, then run gel in electrophoresis tank under 100 V.</p> |
| + | |
| + | <h3>PCR</h3> |
| + | <p> |
| + | Purpose: to check whether our plasmid has successfμlly transformed into E.coli, DH5α.<br> |
| + | 1. PCR condition finding<br> |
| + | * P.S. Prepare the stock solution first, which consist of mQH<sub>2</sub>O、10x buffer、forward primer、backward primer、dNTP and DNA, then add the DNA polymerase before starting the PCR process.<br> |
| + | * 0.5 ~ 1 tube shall be added to the ideal amount when preparing the stock solution.<br> |
| + | It is recommended to dilute the amount polymerase needed to 1 unit/l, to decrease the error of adding small amount of volume.<br><br> |
| + | Recipe |
| + | </p> |
| + | |
| + | <style type="text/css"> |
| + | .tg {border-collapse:collapse;border-spacing:0;border-color:#ccc;} |
| + | .tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#fff;} |
| + | .tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#f0f0f0;} |
| + | .tg .tg-yw4l{vertical-align:top} |
| + | .tg .tg-b7b8{background-color:#f9f9f9;vertical-align:top} |
| + | </style> |
| + | <table class="tg"> |
| + | <tr> |
| + | <th class="tg-yw4l">DNA (10 ng)</th> |
| + | <th class="tg-yw4l">10 ng</th> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8">10x buffer</td> |
| + | <td class="tg-b7b8">5 μl</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-yw4l">forward primer (2.5 μM)</td> |
| + | <td class="tg-yw4l">2.5 μl</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8">Reverse primer (2.5 μM)</td> |
| + | <td class="tg-b7b8">2.5 μl</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-yw4l">dNTP (10 μM)</td> |
| + | <td class="tg-yw4l">1 μl</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8">DNA polymerase (5 unit/μl)</td> |
| + | <td class="tg-b7b8">1~2 unit</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-yw4l">Total volume , add water to 50 μl</td> |
| + | <td class="tg-yw4l">50 μl</td> |
| + | </tr> |
| + | </table> |
| + | |
| + | <br/> |
| + | |
| + | <style type="text/css"> |
| + | .tg {border-collapse:collapse;border-spacing:0;border-color:#ccc;} |
| + | .tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#fff;} |
| + | .tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 5px;border-style:solid;border-width:1px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#f0f0f0;} |
| + | .tg .tg-yw4l{vertical-align:top} |
| + | .tg .tg-b7b8{background-color:#f9f9f9;vertical-align:top} |
| + | </style> |
| + | <table class="tg"> |
| + | <tr> |
| + | <th class="tg-yw4l"></th> |
| + | <th class="tg-yw4l">Final concentration</th> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8">DNA</td> |
| + | <td class="tg-b7b8">10 ng/ 50 μl</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-yw4l">Buffer</td> |
| + | <td class="tg-yw4l">1x</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8">Forward primer</td> |
| + | <td class="tg-b7b8">0.125 μM</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-yw4l">Backward primer</td> |
| + | <td class="tg-yw4l">0.125 μM</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-b7b8">dNTP</td> |
| + | <td class="tg-b7b8">0.2 μM</td> |
| + | </tr> |
| + | <tr> |
| + | <td class="tg-yw4l">DNA polymerase</td> |
| + | <td class="tg-yw4l">1~2 unit</td> |
| + | </tr> |
| + | </table> |
| + | |
| + | <p> |
| + | 2. Condition entering<br> |
| + | a. 94°C for 1 min.<br> |
| + | b. 94°C for 30 sec<br> |
| + | c. The specific temperature for primer to bind on DNA, 30 sec (pSBBS1C 55°C, pSBBS4S 65°C)<br> |
| + | d. 72°C for 1 min<br> |
| + | e. GOTO 2, 25 cycle<br> |
| + | f. 16°C for 10 min (to cool the machine)<br> |
| + | g. Run gel<br> |
| + | h. Result:<br> |
| + | a. pSBBS1C |
| + | |
| + | </p> |
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| + | |
| + | </section> |
| + | |
| + | </article> |
| </div> | | </div> |
| | | |
− | <div class="column half_size">
| + | |
− | <h5>What should this page contain?</h5>
| + | |
− | <ul>
| + | |
− | <li> Protocols </li>
| + | |
− | <li> Experiments </li>
| + | |
− | <li> Documentation of the development of your project </li>
| + | |
− | </ul>
| + | |
| + | |
| + | |
| + | |
| | | |
| </div> | | </div> |
| | | |
− | <div class="column half_size">
| |
− | <h5>Inspiration</h5>
| |
− | <ul>
| |
− | <li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
| |
− | <li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
| |
− | <li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
| |
− | </ul>
| |
| </div> | | </div> |
| | | |
| | | |
− | <div class="clear"></div> | + | <!-- Footer --> |
| + | <div id="footer"> |
| + | <div class="container"> |
| + | <div class="row"> |
| + | <div class="12u"> |
| | | |
| + | <!-- Contact --> |
| + | <section class="contact"> |
| + | |
| + | <ul class="icons"> |
| | | |
− | <div class="column half_size"> | + | <li> |
| + | <a href="https://www.facebook.com/ccuigemteam" target="_blank"> |
| + | <img src="https://static.igem.org/mediawiki/2016/2/2f/T--Harvard_BioDesign--images_facebook01.png"alt="Facebook Logo" style="width:51px;height:51px;"> |
| + | </a> |
| + | </li> |
| | | |
| + | <li> |
| + | <a href="emailto:ccu.igem.2017@gmail.com"> |
| + | <img src="https://static.igem.org/mediawiki/2016/e/e2/T--Harvard_BioDesign--images_gmail01.png" alt="Email Logo" style="width:51px;height:51px;"> |
| + | </a> |
| + | </li> |
| | | |
| | | |
− | </div> | + | |
| + | |
| + | </ul> |
| + | </section> |
| + | |
| + | <!-- Copyright --> |
| + | <div class="copyright"> |
| + | <ul class="menu"> |
| + | <li>© 2017 CCU Taiwan iGEM</li><li>Design: <a href="http://html5up.net">HTML5 UP</a></li> |
| + | </ul> |
| + | |
| + | |
| + | </div> |
| + | |
| + | </div> |
| + | </div> |
| + | </div> |
| + | |
| + | </div> |
| + | |
| | | |
| + | </body> |
| </html> | | </html> |