Biomod/2013/Tianjin/Protocol: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
Line 61: Line 61:
margin:0 auto;
margin:0 auto;
width:960px;
width:960px;
height:200px;
height:100px;
text-align:center;
text-align:center;
/*background-color:#FF6;*/
/*background-color:#FF6;*/

Revision as of 21:24, 25 October 2013

<html>


<style>

  • {margin:0;padding:0;font-family:"微软雅黑","Arial";}

body{ width: 960px; height:auto; margin: 0 auto; font-size:12px; font-family:Arial, Helvetica, sans-serif; background-color:#fcfcfc; }

h1,h2,h3,h4,h5,h6{border:none;}

p {margin:0.5em 0 !important; text-decoration:none; font-family:Arial, Helvetica, sans-serif; font-size:12px; }

.logo-section{ height:220px; width:960px; position:relative; float:left; margin-top:5px; background:url(#) no-repeat; /*background-color:#0F6;*/ }

.content1{ height:50px; width:960px; position:relative; float:left; /*background-color:#F90;*/ }

.text-box{ width:960px; height:auto; margin-top:0px; float:left; position:relative; /*background-color:#96F;*/ }


.vedio{ width:960px; height:300px; position:relative; float:left; /*background-color:#FCC;*/ }

.link{ margin:0 auto; width:960px; height:100px; text-align:center; /*background-color:#FF6;*/ float:left; }

  1. goTopBtn {POSITION: fixed;TEXT-ALIGN: center;LINE-HEIGHT: 30px;WIDTH: 100px;BOTTOM: 35px;HEIGHT: 100px;FONT-SIZE: 12px;RIGHT: 30px;}
  1. content{margin:0;padding:0;height:1000px;border:0px;}
  1. column-content{background-color:#FFF;}
  1. globalWrapper{

width:960px; height:auto; -moz-box-shadow:0px 0px 15px #000; -webkit-box-shadow:0px 0px 15px #000; box-shadow:0px 0px 15px #000; }

/*hidden section*/

.firstHeading{display:none;}

  1. sidebar-main{display:none;}
  2. p-cactions{display:none;}
  3. p-personal{display:none;}

</style>

<!--banner section--> <div class="logo-section">

</div>


<!--content section-->

<style type="text/css"> .sddm{z-index: 30;width: 960px;height:20px;position:relative;float:left;background-color:#303437;position:raletive;margin-left:0px;} .sddm ul{margin-left:0px;} .sddm li{margin: 0;list-style: none;float: left;font: bold 14px arial;height:20px;background-color:#FFF;border-left:#fff thin solid;border-right:#fff thin solid;} .sddm li a{display: block;margin:0;width: 158px;height:20px;background:#303437;color: #FFF;text-align: center;text-decoration: none;} .sddm li a:hover{background:#999;color:#000;} .sddm div{position: absolute;width:158px;z-index:999;visibility: hidden;margin-top:1px;padding: 0;} .sddm div a{position: relative;display: block;margin: 0;padding: 5px 5px;width: auto;white-space: nowrap;text-align:center;text-decoration: none;color: #FFF;font: 12px arial;height:20px;} .sddm div a:hover{background:#999;color:#000;}

</style> <script type="text/javascript"> var timeout = 500; var closetimer = 0; var ddmenuitem = 0;

function mopen(id) { mcancelclosetime(); if(ddmenuitem) ddmenuitem.style.visibility = 'hidden'; ddmenuitem = document.getElementById(id); ddmenuitem.style.visibility = 'visible';} function mclose() { if(ddmenuitem) ddmenuitem.style.visibility = 'hidden';} function mclosetime() { closetimer = window.setTimeout(mclose, timeout);} function mcancelclosetime() { if(closetimer) { window.clearTimeout(closetimer); closetimer = null;}} document.onclick = mclose; </script>


<div class="content1"> <ul class="sddm"> 

   <li><a href="http://openwetware.org/wiki/Biomod/2013/Tianjin" onmouseover="mopen('m1')" onmouseout="mclosetime()" style="padding:15px 0px;">Home</a>

</li> <li><a href="http://openwetware.org/wiki/Biomod/2013/Tianjin/Team" onmouseover="mopen('m2')" onmouseout="mclosetime()" style="padding:15px 0px;">Team</a> 

       <div id="m2" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">

<a href="#">Item1</a> <a href="#">Item2</a> <a href="#">Item3</a> <a href="#">Item4</a> </div> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/Tianjin/Designs" onmouseover="mopen('m3')" onmouseout="mclosetime()" style="padding:15px 0px;">Designs</a> 

       <div id="m3" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">

<a href="#">Item1</a> <a href="#">Item2</a> <a href="#">Item3</a> <a href="#">Item4</a> </div> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/Tianjin/Experiments &amp; Results" onmouseover="mopen('m4')" onmouseout="mclosetime()" style="height:35px;padding:8px 0px 7px 0px;">Experiments &amp; Results</a> 

       <div id="m4" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">

<a href="#">Item1</a> <a href="#">Item2</a> <a href="#">Item3</a> <a href="#">Item4</a> </div> </li> 

   <li><a href="http://openwetware.org/wiki/Biomod/2013/Tianjin/Protocol" onmouseover="mopen('m5')" onmouseout="mclosetime()" style="padding:15px 0px;">Protocol</a>

</li> 

   <li><a href="http://openwetware.org/wiki/Biomod/2013/Tianjin/Attributions" onmouseover="mopen('m6')" onmouseout="mclosetime()" style="padding:15px 0px;">Attributions</a>
       <div id="m6" onmouseover="mcancelclosetime()" onmouseout="mclosetime()">

<a href="#">Item1</a> <a href="#">Item2</a> <a href="#">Item3</a> <a href="#">Item4</a> </div> </li> </ul> </div>


<div class="text-box"> <style type="text/css"> .box{ width:960px;height:auto; margin:0 auto; overflow:hidden;}

.main{ width:710px; height:auto; float:right;position:relative;padding:10px 10px 10px 10px;font-size:12px;} .fixed{ width:220px; height:400px; font:normal; text-align:center;float:left;word-spacing:0.1em;top:10px;margin-top:10px;} .main img{border:hidden;margin-bottom:5px;} .main div,li,p{font-family:Arial;line-height:150%;word-spacing:0.1em;font-size:12px;}


.box p{color:#000;font-family:Arial, Helvetica, sans-serif;font-size:12px;line-height:150%;text-align:left; clear:both;} .box h1{text-decoration:none;font-weight:normal;color:#000;} .img1{margin:0 150px 15px 150px;padding:5px 5px 5px 5px;background-color:#fafafa;border:thin solid #999; vertical-align:middle;width:400px;} .img1 a{target="_blank";}

</style>


<div class="box">

<div class="fixed"> <style>

  1. toc {

width:220px; margin-left:-225px; }

.tocbefore { position:absolute; }

.tocafter { position:fixed; top:30px; }

</style>

<script type="text/javascript"> function getScrollTop() { 

   var scrollPos; 
   if (window.pageYOffset) { 
       scrollPos = window.pageYOffset; 
   } 
   else if (document.compatMode) { 
   	if(document.compatMode != 'BackCompat')
   	{
       	scrollPos = document.documentElement.scrollTop; 
       }
       else if (document.body) 
       { 
       	scrollPos = document.body.scrollTop; 
   	}
   } 
   if(scrollPos < 290){

document.getElementById("toc").className="tocbefore"; } else{ document.getElementById("toc").className="tocafter"; } } window.onscroll=getScrollTop; </script> </div>

<div class="main"> </html>

The DNA—Track

<html>

<hr /><br />

<ul> 

 <li>Version 1
   <ul>
     <li>T1
         <ul>
             <li>5- CTCCCCG CGAAAAG AGTGTAC AGCGAAC GCCACG GTAAGCC TTCTTTC GCTTACC-3</li>
         </ul>
     </li>
     <li>T2
         <ul>
             <li>5- CTCCCCG CGAAAAG AGTGTAC AGCGAAC GCCACG GTAAGCC TTCTTTC GCTTACC-3</li>
             
         </ul>
     </li>
     <li>T3
         <ul>
             <li>5- CTCCCCG CGAAAAG AGTGTAC AGCGAAC GCCACG GTAAGCC TTCTTTC GCTTACC-3</li>
             
         </ul>
     </li>
     <li>T4
         <ul>
             <li>5- CTCCCCG CGAAAAG AGTGTAC AGCGAAC GCCACG GTAAGCC TTCTTTC GCTTACC-3</li>
             
         </ul>
     </li>
     <li>Trigger
         <ul>
             <li>5-CAGCGAACCTTTTCG AAAGCG GGGAG TTGCGTCCGTAT-3</li>

         </ul>
     </li>
   </ul>
 </li>
 <li>Version 2
   <ul>
     <li>T-1: 
         <ul>
             <li>ACGGCGAGGAGGGTTAACCCAGACGCCGAATCCTAGACTCAAAGTAGTCTAGGATTCGGCG<br/>TCTGAAAGGTAAGCCGTAGCTTCTTCTAATACGGCTTACC</li>

         </ul>
     </li>
     <li>T-2: 
         <ul>
             <li>CCATTCGGCATAATCTTCTTCGATGCCGAATGGTTTAGTCTAGGATTCGGCGTCTGGACTCT<br/>CAGACGCCGAATCCTAGACTACTTTGGACAGGAGCGGC</li></ul>

     </li>
     <li>T-3:
         <ul>
             <li>ACGGCTTACCTTTAGAGTCCAGACGCCGAATCCTAGACTGGACTTAGTCTAGGATTCGGCGT<br/>CTGCCCTCCTCGCCGTCAGCTTTCTCTAATACGGCGAGGA</li>

         </ul>
     </li>
     <li>T-4: 
         <ul>
             <li>AGGAGCGGCATAATCTCTTTCGATGCCGCTCCTGTCAGTCTAGGATTCGGCGTCTGAATTGG<br/>CAGACGCCGAATCCTAGACTAAGTCCAAACCATTCGGCA</li>
         </ul>
     </li>
     <li>Trigger:
         <ul>
             <li>AGTCTAGGATTCGGCGTCTG&nbsp;GGTTAA</li></ul>
     </li>
   </ul>
 </li>
 <li>Version 3
   <ul>
     <li>T1
         <ul>
             <li>5-ATACGGACGCAACTCCCCGCTTTCGAAAAGGTTCGCTGAGTGTACAGCGAACCTTTTCGG<br/>CCACGGTAAGCCGTATAGCTTCTTTCTAATACGGCTTACC-3</li>
         </ul>
     </li>
     <li>T2
         <ul>
             <li>5-GGTAAGCCGTATAGCTTCTTTCTAATACGGCTTACCGGTACCAGCGAACCTTTTCGAAAGC<br/>G&nbsp;CGAAAAGGTTCGCTGTACACTCACGTTTGCGTCCGTAT-3</li>
         </ul>
     </li>
     <li>T3
         <ul>
             <li>5-ATACGGCTTACCGTGGCCGCTTTCGAAAAGGTTCGCTGAGTGTACAGCGAACCTTTTCGG<br/>GGAG TTGCGTCCGTATAGCTCTTCTCTAATACGGACGCAA-3</li>
         </ul>
     </li>
     <li>T4
         <ul>
             <li>5-TTGCGTCCGTATAGCTCTTCTCTAATACGGACGCAAACGTGCAGCGAACCTTTTCGAAAG<br/>CG CGAAAAGGTTCGCTGTACACTGTACCGGTAAGCCGTAT-3</li>
         </ul>
     </li>
     <li>Trigger
         <ul>
             <li>5-CAGCGAACCTTTTCG AAAGCG GGGAG TTGCGTCCGTAT-3</li>
            
         </ul>
     </li>
   </ul>
 </li>

</ul> </html>

The Walker

<html>

<hr /><br /> <ul> 

 <li>Quadruped-walker
   <ul>
     <li>WALKER1 
         <ul>
             <li>5-ATGGACTACGCTACCATTTTTTTTTTTTTTTTTTTTGGTAAGCCTGGGCCTCTTTCTTTTTA<br/>AGAAAGAAC-3</li>
         </ul>
     </li>
     <li>WALKER2
         <ul>
             <li>5-TATAGCACGGTAAACTTAGTCCATTTTTTTTTTTTTTTTTTTTTGGTAAGCCTGGGCCTCTT<br/>TCTTTTTAAGAAAGAAC-3</li>
             
         </ul>
     </li>
     <li>WALKER3
         <ul>
             <li>5-TGGTAGCGAGTTTACCTTACGGAGTTTTTTTTTTTTTTTTTTTTTTGCGTCCTGGGCCTCT<br/>CTTCTTTTAGAAGAGAAC-3</li>
             
         </ul>
     </li>
     <li>WALKER4
         <ul>
             <li>5-CTCCGTAACTCGTATATTTTTTTTTTTTTTTTTTTTTTGCGTCCTGGGCCTCTCTTCTTTT<br/>AGAAGAGAAC-3</li>
         </ul>
     </li>
     <li>Two-foot-walker</li>
   </ul>
 </li>
 <li>W2KER1
   <ul>
     <li>5-TTTCACATGCAATCGAACGTTTTTTTTTTTTGGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGA<br/>AC-3</li>
   </ul>
 </li>
 <li>W2KER2 
   <ul>
    <li>5-AACGTTCGATTGCATGTGAAATTTTTTTTTTTTGCGTCCTGGGCCTCTCTTCTTTTAGAAGAG<br/>AAC-3</li>
   </ul>
 </li>

</ul>


</html>

Agarose electrophoresis

<html>

<hr /><br /> <ul> <li>0.2g agarose mixed with 20ml 1X TAE, heat to boil </li> <li> When temperature drops, add 0.1μL golden view, cooling for 30min </li> <li> Adding 1X TAE into </li> <li> Mix 2μL 6X loading buffer with 5μL DNA sample, add 7μL of the sample into the well </li> <li> Start the electrophoresis at 150-250V, for 10-30min, depending on the blue band of loading buffer </li> <li> Stop the electrophoresis, put the gel into UV gel imager, expose for 80ms </li> </ul>

<br/> </html>

PAGE

<html>

<hr /><br /> <p> Nucleic acid electrophoresis utilizes the same concepts as protein electrophoresis. The denaturant and visualization techniques differ. All nucleic acids are negatively charged. It is not neccessary or desirable to use a charged denaturant such as SDS. Urea is used to denature the DNA or RNA within the gel. Visualization is usually achieved via staining with ethidium bromide. Ethidium bromide intercalates between base pairs and fluoresces. The fluoresence of free (non-bound) ethidium bromide is quenched. Note that ethidium bromide is a carcinogen and that UV light can damage skin and eyes. Gloves and protective eyeware should be used. The UV box has special anti-UV plastic and a safety interlock. Do not try to defeat the interlock.</p>

</html>

Materials

<html>


<p> Glass plates (10 x 20 cm), spacers, comb, and clamps</p> <p> Power supply</p> <p> Nucleic Acid samples </p> <p>6.66 mL 30% Acrylamide </p> <p>3.34 mL 5X TBE </p> <p>8.41 g Urea </p>


<p> The TA will have prepared the solution above before lab starts. The solution must be heated to dissolve the urea. However warm gel solutions polymerize quickly with unpredictable results, so the solution must be allowed to cool before use.</p>


<p>140 microliters 10% Ammonium Persulfate</p> <p>7 microliters TEMED</p> <p>Loading buffer- 2X Dye- 0.25% Bromophenol Blue, 0.25% Xylene Cyanol</p> <p>Gel Red</p> <p>1X TBE for Running Buffer</p>

</html>

Methods

<html>


<p> Very similar to SDS PAGE gel. You should be able to assemble gel by yourself. Have the TA inspect gel prior to use.</p> <p> Add 8 microliters of the Loading Buffer to 10 microliters of your nucleic acid sample Load onto gel, and separate your nucleic acid fragments by electrophoresis at around 30 Amps, until the dye front approaches the bottom of gel. Do NOT let dye front run off gel.</p> <p Stain with Gel Red for 20 minutes. Visualize on the UV box. Caution: Wear safety glasses around UV light.</p> <p> Photograph the gel. Do not dispose of the gel until you have ensured that you have a reasonable photograph.</p> </br> </html>

PCR Annealing Procedure (for Logic Gate)

<html> <hr /><br />

<ul> <li>90℃ for 5 min;</li> <li>90℃ for 1min;-1℃ per cycle</li> <li> Go to 2 for 86 times </li> <li>4℃ forever</li> </ul> <br/> </html>

Recipe

<html> <hr /><br />

<ul> <li> DNAzyme Buffer</br> <p>1.Inoculate 5μl BL21(Glycerol Storage) into 3 ml LB medium for an overnight cultures at 37 ℃ with 220rpm shaking</p></li> <li> TBE</br> <p>10x TBE (1 liter): Dissolve 108 g Tris and 55 g Boric acid in 900 ml distilled water. Add 40 ml 0.5 M Na2EDTA (pH 8.0) (alternatively use 9.3 g Na2EDTA) Adjust volume to 1 Liter. Store at room temperature. Note: 10x TBE may take some time to dissolve, even with fast stirring TBE can be diluted to 1X prior to use in electrophoresis, 0.5x is acceptable as well.</p></li> <li> TAE</br> <p> TAE buffer is commonly prepared as a 50X stock solution for laboratory use. A 50X stock solution can be prepared by dissolving 242g Tris base in water, adding 57.1mL glacial acetic acid, and 100mL of 500mM EDTA (pH 8.0) solution, and bringing the final volume up to 1 liter. This stock solution can be diluted 50:1 with water to make a 1X working solution. This 1X solution will contain 40mM Tris, 20mM acetic acid, and 1mM EDTA. </p></li> <li> Urea Loading Buffer </br> <p>8 M urea, 50 mM EDTA, 0.05% xylene cyanol, and 50 mM Tris acetate, pH 8.2 </p></li>

</div>


</div>

<div class="link"> <p><br /> <br /> E-mail：austinamens@gmail.com | Address:Building No.20, No.92 Weijin Road, Tianjin, China | Zip-code：300072 <br />Copyright 2013 © Tianjin University iGEM Team</p> </div>


</div>


<!--<div class="vedio">

</div>-->


<!--top button section--> <div style="display: block" id="goTopBtn"> <a href="#top" title="Top"><img border=0 src="#"></a> </div>


<a href="http://openwetware.org/index.php?title=Biomod/2013/Tianjin/Protocol&action=edit">edit</a>





</html>

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2013/Tianjin/Protocol&oldid=746262"