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− | @media screen and (max-width: | + | @media screen and (max-width: 992px) { |
#section1, #section2, #section3, #section4, #section5 { | #section1, #section2, #section3, #section4, #section5 { | ||
margin-left: 50px; background-color: #eee; color:#171717; | margin-left: 50px; background-color: #eee; color:#171717; | ||
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<div id="section1" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;"> | <div id="section1" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;"> | ||
<h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Introduction</h3> | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Introduction</h3> | ||
− | <p style="padding:10px">It is significant for synthetic biology to develop a reliable and repeatable measurement, the same to all the other engineering disciplines. We HUST-China have volunteered to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable by | + | <p style="padding:10px">It is significant for synthetic biology to develop a reliable and repeatable measurement, the same to all the other engineering disciplines. We HUST-China have volunteered to test some RBS devices (BCDs) that are intended to make gene expression more precise and reliable by measure the expression level of GFP, in order to help the iGEM community collect data about how reliable will these devices turn out to be in labs around the world. |
</p> | </p> | ||
</div> | </div> | ||
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<tr> | <tr> | ||
<td>Haibo Huang </td> | <td>Haibo Huang </td> | ||
− | <td> | + | <td>u201512127@hust.edu.cn</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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<h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Chassis and Safety</h3> | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Chassis and Safety</h3> | ||
<h4><strong> What chassis did you use?</strong> </h4> | <h4><strong> What chassis did you use?</strong> </h4> | ||
− | <p>Escherichia coli | + | <p>Escherichia coli DH5alpha</p> |
<h4><strong> What Biosafety Level is your chassis? </strong> </h4> | <h4><strong> What Biosafety Level is your chassis? </strong> </h4> | ||
<p>BSL1</p> | <p>BSL1</p> | ||
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<h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Instrument</h3> | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Instrument</h3> | ||
<h4><strong> What instrument did you use during your measurements? </strong> </h4> | <h4><strong> What instrument did you use during your measurements? </strong> </h4> | ||
− | <p> | + | <p>plate reader</p> |
<h4><strong> Please provide the brand and model of your instrument.</strong> </h4> | <h4><strong> Please provide the brand and model of your instrument.</strong> </h4> | ||
<p>Flexstation 3</p> | <p>Flexstation 3</p> | ||
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<li>②Add 100 μl LUDOX 100 % into wells A1, B1, C1, D1</li> | <li>②Add 100 μl LUDOX 100 % into wells A1, B1, C1, D1</li> | ||
<li>③Add 100 μl of dH2O into A2, B2, C2, D2</li> | <li>③Add 100 μl of dH2O into A2, B2, C2, D2</li> | ||
− | <li>④Measure absorbance 600 nm of all samples in all standard measurement | + | <li>④Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</li> |
+ | <li>⑤Import data into "Abs600" blue cells in provided Excel calibration sheet</li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
− | <h4><strong> B1. Protocol for | + | <h4><strong> B1. Protocol for FIuorescein Fluoresence standard curve</strong> </h4> |
<h5><strong>Did you use pathlength correction during measurement? </strong> </h5> | <h5><strong>Did you use pathlength correction during measurement? </strong> </h5> | ||
<p>Yes</p> | <p>Yes</p> | ||
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<li>③ Dilute the 2x fluorescein stock solution using 1xPBS to make a 1x fluorescein solution (final concentration is 50 μM).</li> | <li>③ Dilute the 2x fluorescein stock solution using 1xPBS to make a 1x fluorescein solution (final concentration is 50 μM).</li> | ||
<li>④ *Illustration of serial dilution samples in 96 well plate or cuvettes: value decreases by 2-fold with each column (50% in column 2, 25% in column 3, 12.5% in column 4, etc.)</li> | <li>④ *Illustration of serial dilution samples in 96 well plate or cuvettes: value decreases by 2-fold with each column (50% in column 2, 25% in column 3, 12.5% in column 4, etc.)</li> | ||
+ | <li>⑤Import data into "Abs600" blue cells in provided Excel calibration sheet</li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
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<li>⑫ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.</li> | <li>⑫ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11.</li> | ||
<li>⑬ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.</li> | <li>⑬ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.</li> | ||
− | <li>⑭ | + | <li>⑭ TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.</li> |
<li>⑮ Repeat dilution series for rows B, C, D. | <li>⑮ Repeat dilution series for rows B, C, D. | ||
<li>⑯ Measure fluorescence of all samples in all standard measurement modes in instrument. | <li>⑯ Measure fluorescence of all samples in all standard measurement modes in instrument. | ||
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<li class="dropdown" style="list-style-type:none;"> | <li class="dropdown" style="list-style-type:none;"> | ||
<button href="#" class="dropdown-toggle" data-toggle="dropdown"> | <button href="#" class="dropdown-toggle" data-toggle="dropdown"> | ||
+ | More details <b class="caret"></b> | ||
+ | </button> | ||
+ | <ul class="dropdown-menu" style="padding: 10px; background:#ccc;"> | ||
+ | |||
+ | <li>① Set your instrument to read Abs600 (as OD calibration setting)</li> | ||
+ | <li>② Measure Abs600 of the overnight cultures</li> | ||
+ | <li>③ Import data into blue cells in Excel Dilultion Calculation sheets provided</li> | ||
+ | <li>④ Dilute the cultures to a target Abs600 of 0.02 (see the volume of preloading culture and media in Excel Dilution Calculation sheets) in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube.</li> | ||
+ | <li>⑤ Remove 500uL of each culture for your zero time point and hold these samples on ice. (You should have 16 sample tubes from the time=0 time point)</li> | ||
+ | <li>⑥ Incubate the cultures at 37°C and 220 rpm.</li> | ||
+ | <li>⑦ Remove 500uL samples of each culture after 2, 4 and 6 hours of growth. Keep samples on ice while completing the additional time points. You should have 16 sample tubes for each time point.</li> | ||
+ | <li>⑧ Set up your four measurement plates: For colony #1 culture, pipet 100 uL samples into wells A, B, C and D of the row for that device. For colony #2, pipet 100uL samples into wells E, F, G, and H of the row for that device.</li> | ||
+ | <li>⑨ Read your plates, taking care to use the exact same settings used for your fluorescein measurement.</li> | ||
+ | |||
+ | |||
+ | </ul> | ||
+ | </li> | ||
+ | <h5><strong>The initial OD600 measurement of our overnight cultures.</strong> </h5> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/1/1a/2017_HUST_China_interlab_img_table1.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | </div> | ||
+ | <div class="col-xs-12"> | ||
+ | <h5><strong>What type of media did you use for this step? </strong> </h5> | ||
+ | <p>Luria Bertani </p> | ||
+ | <h5><strong>What type of vessel or container did you use to grow your cells? </strong> </h5> | ||
+ | <p>50 ml Falcon tube</p> | ||
+ | <h5><strong>What temperature setting did you use during the measurement?</strong> </h5> | ||
+ | <p>22℃</p> | ||
+ | <h5><strong>What type of 96-well plates did you use?</strong> </h5> | ||
+ | <p></p> | ||
+ | <h5><strong>Black plates with transparent/clear bottom (preferred)</strong> </h5> | ||
+ | <p>Flat</p> | ||
+ | <h5><strong>Measurement </strong> </h5> | ||
+ | <ul class="yuandian"> | ||
+ | <li>Measure OD and fluorescence of all samples</li> | ||
+ | <li>Import data into blue cells in Excel (cell measurement) sheets provided</li> | ||
+ | </ul> | ||
+ | <h5><strong>Suggested Plate Layout for 96-well Plate</strong> </h5> | ||
+ | <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/> | ||
+ | <a href="https://static.igem.org/mediawiki/2017/8/87/2017_HUST_China_interlab_img_003.png" rel="lightbox-demo" title="my caption"><br /> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/8/87/2017_HUST_China_interlab_img_003.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | </a> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="section7" style="border: solid 1px #666; margin:5px; padding:5px;border-radius:10px;overflow: hidden;"> | ||
+ | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px;margin-top:0;">Interlab Resultn</h3> | ||
+ | <h5 class="col-xs-12"><strong>OD600 Reference Point</strong> </h5> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/5/52/2017_HUST_China_interlab_img_table2.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | <h5 class="col-xs-12"><strong>Fluorescence standard curve</strong> </h5> | ||
+ | <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/> | ||
+ | <a href="https://static.igem.org/mediawiki/2017/a/a1/2017_HUST_China_interlab_img_table3.png" rel="lightbox-demo" title="my caption"><br /> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/a/a1/2017_HUST_China_interlab_img_table3.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | </a> | ||
+ | <p class="col-xs-12"><strong>NOTE: 50uM Sample exceeds the range of measurements</strong> </p> | ||
+ | <h5 class="col-xs-12"><strong>OD600 Reference Point</strong> </h5> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/d/d1/2017_HUST_China_interlab_img_table4.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/d/df/2017_HUST_China_interlab_img_table5.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/> | ||
+ | <a href="https://static.igem.org/mediawiki/2017/b/b3/2017_HUST_China_interlab_img_table6.png" rel="lightbox-demo" title="my caption"><br /> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/b/b3/2017_HUST_China_interlab_img_table6.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | </a> | ||
+ | <p class="col-xs-12"><strong>Final scaling level determined from medium-high points likely to be less impacted by saturation or pipetting error.</strong> </p> | ||
+ | <p class="col-xs-12"><strong>If needed, you can shift which points are used, but it is likely better to correct instrument settings and protocol.</strong> </p> | ||
+ | <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/> | ||
+ | <a href="https://static.igem.org/mediawiki/2017/b/b8/2017_HUST_China_interlab_img_table7.png" rel="lightbox-demo" title="my caption"><br /> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/b/b8/2017_HUST_China_interlab_img_table7.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | </a> | ||
+ | <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/> | ||
+ | <a href="https://static.igem.org/mediawiki/2017/e/ea/2017_HUST_China_interlab_img_table8.png" title="my caption"><br /> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/e/ea/2017_HUST_China_interlab_img_table8.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | </a> | ||
+ | <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/> | ||
+ | <a href="https://static.igem.org/mediawiki/2017/d/d2/2017_HUST_China_interlab_img_table9.png" rel="lightbox-demo" title="my caption"><br /> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/d/d2/2017_HUST_China_interlab_img_table9.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | </a> | ||
+ | <p><strong><span class="col-xs-12">Click on the picture and you can see a clearer picture.Then click on the return key of the browser and you can return.</span></strong></p><br/> | ||
+ | <a href="https://static.igem.org/mediawiki/2017/2/2b/2017_HUST_China_interlab_img_table10.png" rel="lightbox-demo" title="my caption" style="padding: 10px;"><br /> | ||
+ | <img title="demo1" src="https://static.igem.org/mediawiki/2017/2/2b/2017_HUST_China_interlab_img_table10.png" alt="demo1" class="col-xs-8 col-xs-offset-2"> | ||
+ | </a> | ||
+ | |||
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Revision as of 20:08, 31 October 2017