Team:XMU-China/Content5

From 2013.igem.org

(Difference between revisions)
 
(38 intermediate revisions not shown)
Line 10: Line 10:
<link rel="stylesheet" type="text/css" href="https://2013.igem.org/Team:XMU-China/style2.css?action=raw&ctype=text/css" >
<link rel="stylesheet" type="text/css" href="https://2013.igem.org/Team:XMU-China/style2.css?action=raw&ctype=text/css" >
<link rel='stylesheet' type='text/css' href='http://fonts.googleapis.com/css?family=Oswald' >
<link rel='stylesheet' type='text/css' href='http://fonts.googleapis.com/css?family=Oswald' >
-
 
+
<style>
 +
table,td,tr,th{background-color:transparent;cellspacing=0px;}</style>
</head>
</head>
-
<body>
+
<body style="font-family:"Times New Roman", Times, serif">
 +
<!-- START MENU SECTION -->
 +
<!-- end #menu-bar -->
 +
<!-- END MENU SECTION -->
 +
 
 +
 
 +
<!-- START LOGO SECTION -->
 +
<div id="logo-bar"><!-- .center-block -->
 +
</div><!-- #logo-bar -->
 +
<!-- END LOGO SECTION -->
<!-- START WRAPPER SECTION -->
<!-- START WRAPPER SECTION -->
<div id="wrapper">
<div id="wrapper">
Line 41: Line 51:
<li>3. Protein electrophoresis</li>
<li>3. Protein electrophoresis</li>
<h4>3 Microfluidic Protocol</h4>
<h4>3 Microfluidic Protocol</h4>
-
<li>1. Pre-procession of Dh5a</li>
+
<li>1. Pre-procession of DH5α</li>
<li>2. Pre-procession of LB</li>
<li>2. Pre-procession of LB</li>
<li>3. Wetting the chip & Capturing images</li>
<li>3. Wetting the chip & Capturing images</li>
-
                <h4>4 SDS-PAGE</h4>
 
-
<li>1. Preparation
 
-
<li>2. Bacteria culture</li>
 
-
<li>3. Protein electrophoresis</li>
 
-
 
</div><!-- .center-block-page -->
</div><!-- .center-block-page -->
Line 64: Line 69:
<div id="separator">
<div id="separator">
<div class="center-block">
<div class="center-block">
-
<a name="Lecture"></a>
+
<a name="Circuit_Construction"></a>
-
<h3>Circuit Construction</h3>
+
<h3>1. Circuit Construction</h3>
</div>
</div>
</div>
</div>
Line 76: Line 81:
<p>
<p>
-
   LB medium: 5g Yeast Extract, 10g Tryptone and 10g NaCl, add ddH2O to 1L.<br/>
+
   LB medium: 5g Yeast Extract, 10g Tryptone and 10g NaCl, add ddH<SUB>2</SUB>O to 1L.<br/>
-
   LB solid medium: 5g Yeast Extract, 10g Tryptone, 10g NaCl and 15 g agar, add ddH2O to 1L.<br/>
+
   LB solid medium: 5g Yeast Extract, 10g Tryptone, 10g NaCl and 15 g agar, add ddH<SUB>2</SUB>O to 1L.<br/>
</p><br/>
</p><br/>
<b>1.2 Antibiotic</b>
<b>1.2 Antibiotic</b>
Line 93: Line 98:
     <td>100 mg/ml</td>
     <td>100 mg/ml</td>
     <td>50 ug/ml</td>
     <td>50 ug/ml</td>
-
     <td>ddH2O</td>
+
     <td>ddH<SUB>2</SUB>O</td>
     <td>filtration</td>
     <td>filtration</td>
Line 115: Line 120:
     <td>25 mg/ml</td>
     <td>25 mg/ml</td>
     <td>25 ug/ml</td>
     <td>25 ug/ml</td>
-
     <td>ddH2O</td>
+
     <td>ddH<SUB>2</SUB>O</td>
     <td>filtration</td>
     <td>filtration</td>
   </tr>
   </tr>
Line 201: Line 206:
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td><i>E. coli</i> DH5a</td>
+
     <td><i>E. coli</i> DH5α</td>
     <td>cloning</td>
     <td>cloning</td>
     <td>Lab stock</td>
     <td>Lab stock</td>
Line 226: Line 231:
<tr>
<tr>
<td width="105" rowspan="2" >
<td width="105" rowspan="2" >
-
<p class="MsoListParagraph">
+
<a name="_Hlk367271772"></a><span style="font-size:10.0pt;font-family:&quot;">Part</span>
<a name="_Hlk367271772"></a><span style="font-size:10.0pt;font-family:&quot;">Part</span>
-
</p>
+
</td>
</td>
<td width="90" rowspan="2" >
<td width="90" rowspan="2" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">Backbone</span>
<span style="font-size:10.0pt;font-family:&quot;">Backbone</span>
-
</p>
+
</td>
</td>
<td width="142" rowspan="2" >
<td width="142" rowspan="2" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">Type</span>
<span style="font-size:10.0pt;font-family:&quot;">Type</span>
-
</p>
+
</td>
</td>
<td width="99" rowspan="2" >
<td width="99" rowspan="2" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">Location</span>
<span style="font-size:10.0pt;font-family:&quot;">Location</span>
-
</p>
+
</td>
</td>
<td width="161" colspan="2" >
<td width="161" colspan="2" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">Size (bp)</span>
<span style="font-size:10.0pt;font-family:&quot;">Size (bp)</span>
-
</p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<b><span style="font-size:10.0pt;font-family:&quot;">part</span></b>
<b><span style="font-size:10.0pt;font-family:&quot;">part</span></b>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<b><span style="font-size:10.0pt;font-family:&quot;">backbone</span></b>
<b><span style="font-size:10.0pt;font-family:&quot;">backbone</span></b>
-
</p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td width="105" >
<td width="105" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">BBa_K546000</span>
<span style="font-size:10.0pt;font-family:&quot;">BBa_K546000</span>
-
</p>
+
</td>
</td>
<td width="90" >
<td width="90" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:#282828;background:white;">pSB1C3</span>
<span style="font-size:10.0pt;font-family:&quot;color:#282828;background:white;">pSB1C3</span>
-
</p>
+
</td>
</td>
<td width="142" >
<td width="142" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:#282828;background:white;">Signaling</span><span style="font-size:10.0pt;font-family:&quot;"></span>
<span style="font-size:10.0pt;font-family:&quot;color:#282828;background:white;">Signaling</span><span style="font-size:10.0pt;font-family:&quot;"></span>
-
</p>
+
</td>
</td>
<td width="99" >
<td width="99" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2013-P1-12D</span>
<span style="font-size:10.0pt;font-family:&quot;">2013-P1-12D</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
        <span style="font-size:10.0pt;font-family:&quot;">1964</span>
-
<span style="font-size:10.0pt;font-family:&quot;">1964</span>
+
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2070</span>
<span style="font-size:10.0pt;font-family:&quot;">2070</span>
-
</p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td width="105" >
<td width="105" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">BBa_I763020</span>
<span style="font-size:10.0pt;font-family:&quot;">BBa_I763020</span>
-
</p>
+
</td>
</td>
<td width="90" >
<td width="90" >
-
<p class="MsoNormal" align="center" style="text-align:">
+
<span style="font-size:10pt;font-family:'Times New Roman', serif;color:#282828;">pSB1C3</span><span style="font-size:10.0pt;font-family:&quot;color:#282828;"></span>
<span style="font-size:10pt;font-family:'Times New Roman', serif;color:#282828;">pSB1C3</span><span style="font-size:10.0pt;font-family:&quot;color:#282828;"></span>
-
</p>
+
</td>
</td>
<td width="142" >
<td width="142" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:#282828;">Intermediate</span>
<span style="font-size:10.0pt;font-family:&quot;color:#282828;">Intermediate</span>
-
</p>
+
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:#282828;">RBS-GFP-TT</span>
<span style="font-size:10.0pt;font-family:&quot;color:#282828;">RBS-GFP-TT</span>
-
</p>
+
</td>
</td>
<td width="99" >
<td width="99" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2013-P3-11H</span>
<span style="font-size:10.0pt;font-family:&quot;">2013-P3-11H</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">914</span>
<span style="font-size:10.0pt;font-family:&quot;">914</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2070</span>
<span style="font-size:10.0pt;font-family:&quot;">2070</span>
-
</p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td width="105" >
<td width="105" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">BBa_F2621</span>
<span style="font-size:10.0pt;font-family:&quot;">BBa_F2621</span>
-
</p>
+
</td>
</td>
<td width="90" >
<td width="90" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:#282828;background:white;">pSB1A2</span>
<span style="font-size:10.0pt;font-family:&quot;color:#282828;background:white;">pSB1A2</span>
-
</p>
+
</td>
</td>
<td width="142" >
<td width="142" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:#282828;background:white;">Signaling</span><span style="font-size:10.0pt;font-family:&quot;"></span>
<span style="font-size:10.0pt;font-family:&quot;color:#282828;background:white;">Signaling</span><span style="font-size:10.0pt;font-family:&quot;"></span>
-
</p>
+
</td>
</td>
<td width="99" >
<td width="99" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2013-P2-21F</span>
<span style="font-size:10.0pt;font-family:&quot;">2013-P2-21F</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">1158</span>
<span style="font-size:10.0pt;font-family:&quot;">1158</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2079</span>
<span style="font-size:10.0pt;font-family:&quot;">2079</span>
-
</p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td width="105" >
<td width="105" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">BBa_K546001</span>
<span style="font-size:10.0pt;font-family:&quot;">BBa_K546001</span>
-
</p>
+
</td>
</td>
<td width="90" >
<td width="90" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;background:white;">pSB1C3</span><span style="font-size:10.0pt;font-family:&quot;"></span>
<span style="font-size:10.0pt;font-family:&quot;background:white;">pSB1C3</span><span style="font-size:10.0pt;font-family:&quot;"></span>
-
</p>
+
</td>
</td>
<td width="142" >
<td width="142" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">Device</span>
<span style="font-size:10.0pt;font-family:&quot;">Device</span>
-
</p>
+
</td>
</td>
<td width="99" >
<td width="99" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2013-P1-12F</span>
<span style="font-size:10.0pt;font-family:&quot;">2013-P1-12F</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2135</span>
<span style="font-size:10.0pt;font-family:&quot;">2135</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2070</span>
<span style="font-size:10.0pt;font-family:&quot;">2070</span>
-
</p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td width="105" >
<td width="105" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">Ba_J04450</span>
<span style="font-size:10.0pt;font-family:&quot;">Ba_J04450</span>
-
</p>
+
</td>
</td>
<td width="90" >
<td width="90" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">pSB4K5</span>
<span style="font-size:10.0pt;font-family:&quot;">pSB4K5</span>
-
</p>
+
</td>
</td>
<td width="142" >
<td width="142" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">Reporter</span>
<span style="font-size:10.0pt;font-family:&quot;">Reporter</span>
-
</p>
+
</td>
</td>
<td width="99" >
<td width="99" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2013-P5-5G</span>
<span style="font-size:10.0pt;font-family:&quot;">2013-P5-5G</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">1429</span>
<span style="font-size:10.0pt;font-family:&quot;">1429</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:red;">3409</span>
<span style="font-size:10.0pt;font-family:&quot;color:red;">3409</span>
-
</p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td width="105" >
<td width="105" >
-
<p class="MsoNormal">
+
<a name="OLE_LINK5"></a><span style="font-size:10.0pt;font-family:&quot;">BBa_J04450</span><span style="font-size:10.0pt;font-family:&quot;"></span>
<a name="OLE_LINK5"></a><span style="font-size:10.0pt;font-family:&quot;">BBa_J04450</span><span style="font-size:10.0pt;font-family:&quot;"></span>
-
</p>
+
</td>
</td>
<td width="90" >
<td width="90" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">pSB3T5</span>
<span style="font-size:10.0pt;font-family:&quot;">pSB3T5</span>
-
</p>
+
</td>
</td>
<td width="142" >
<td width="142" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">Reporter</span>
<span style="font-size:10.0pt;font-family:&quot;">Reporter</span>
-
</p>
+
</td>
</td>
<td width="99" >
<td width="99" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">2013-P5-7C</span>
<span style="font-size:10.0pt;font-family:&quot;">2013-P5-7C</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">1429</span>
<span style="font-size:10.0pt;font-family:&quot;">1429</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:red;">3241</span>
<span style="font-size:10.0pt;font-family:&quot;color:red;">3241</span>
-
</p>
+
</td>
</td>
</tr>
</tr>
<tr>
<tr>
<td width="105" >
<td width="105" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
-
</p>
+
</td>
</td>
<td width="90" >
<td width="90" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
-
</p>
+
</td>
</td>
<td width="142" >
<td width="142" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
-
</p>
+
</td>
</td>
<td width="99" >
<td width="99" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;color:red;">2013-P3-3H</span><span style="font-size:10.0pt;font-family:&quot;"></span>
<span style="font-size:10.0pt;font-family:&quot;color:red;">2013-P3-3H</span><span style="font-size:10.0pt;font-family:&quot;"></span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
-
</p>
+
</td>
</td>
<td width="81" >
<td width="81" >
-
<p class="MsoNormal">
+
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
-
</p>
+
</td>
</td>
</tr>
</tr>
</tbody>
</tbody>
</table>
</table>
-
<div class="clear"></div>
+
<h4>2. PCR Polymerase Chain Reaction (PCR)</h4>
-
 
+
<br/>
-
+
<b>2.1 General PCR</b>
-
</div><!-- .center-block-page -->
+
<p>Reaction system: </p>
-
<!-- END CONTENT -->
+
<table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="181" style="width:135.95pt;border:none;">
-
</div><!-- #wrapper -->
+
<tbody>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">Template</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">1 μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">Forward Primer</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">2 μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">Reverse Primer</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">2 μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">dNTP Mixture</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">8 μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">10*Ex Buffer</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">5 μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">Ex Taq</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">0.5 μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">ddH<sub>2</sub>O</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">31.5 μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">Total Volume</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">50 μl</span>
 +
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<br/>
 +
<p> Reaction condition:</p>
 +
<table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" align="left" width="500" >
 +
<tbody>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<a name="OLE_LINK2"></a><a name="OLE_LINK1"></a><span style="font-family:&quot;">94 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">4 min</span>
 +
 +
</td>
 +
<td width="79" valign="top" >
 +
 +
<span style="font-family:&quot;">&nbsp;</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">94<sup> o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">1 min</span>
 +
 +
</td>
 +
<td width="79" rowspan="3" >
 +
 +
<span style="font-family:&quot;">30 cycles</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">55 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">0.5 min</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">72 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">1.5 min</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">72 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">10 min</span>
 +
 +
</td>
 +
<td width="79" valign="top" >
 +
 +
<span style="font-family:&quot;">&nbsp;</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
-
<!-- END WRAPPER -->  
+
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">4 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:宋体;">∝</span><span style="font-family:&quot;"></span>
 +
 +
</td>
 +
<td width="79" valign="top" >
 +
 +
<span style="font-family:&quot;">&nbsp;</span>
 +
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
-
<div id="wrapper2">
+
<div class="clear"></div>
-
<style>
+
<b>2.2 Fusion PCR</b>
-
#wrapper2 {background:#ffdcb2}
+
<p>Every biobrick has four sites: EcoR I, Xba I, Spe I and Pst I. When two parts were ligated after digestion by Xba I and Spe I, an 8 bp-size fragment (TACTAGAG) will appear between two parts. If we want to add a fragment in the end of a CDS, for example a fast-degradation tag LVA, the additional 8 bp-size fragment will affect the expression of the CDS. Fusion PCR will resolve this problem. Firstly, two pair primers are designed: F1 and R1 for gene A, F2 and R2 for gene B. The 5'-end of R1 and F2 have about 21-bp homologous region. Secondly, gene A and gene B are amplified via PCR independently. Thirdly, the purified gene A and gene B are fused together via PCR with F1 and R2.</p>
-
</style>
+
<br/>
-
<div id="separator">
+
<b>2.3 Colony PCR</b>
-
<div class="center-block">
+
<p>Reaction system:</p>
-
<a name="Campus_Fair"></a>
+
<table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" align="left" width="181" style="width:135.95pt;border:none;">
-
<h3>Campus Fair</h3>
+
<tbody>
-
+
<tr>
-
</div>
+
<td width="115" valign="top" >
-
</div>
+
 +
<span style="font-family:&quot;">Template</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">colony</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">Forward
 +
  Primer</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">1
 +
  μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">Reverse
 +
  Primer</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">1
 +
  μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">dNTP
 +
  Mixture</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">4
 +
  μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">10*PCR
 +
  Buffer</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">2.5
 +
  μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">rTaq</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">0.25
 +
  μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">ddH<sub>2</sub>O</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">16.25 μl</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="115" valign="top" >
 +
 +
<span style="font-family:&quot;">Total
 +
  Volume</span>
 +
 +
</td>
 +
<td width="66" valign="top" >
 +
 +
<span style="font-family:&quot;">25
 +
  μl</span>
 +
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
-
<!-- START CONTENT -->
+
<div class="clear"></div>
-
<div class="center-block-page clearfix">
+
<p>Reaction condition:</p>
 +
<table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" align="left" width="206" style="width:154.55pt;border:none;">
 +
<tbody>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">94 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">4 min</span>
 +
 +
</td>
 +
<td width="79" valign="top" >
 +
 +
<span style="font-family:&quot;">&nbsp;</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">94<sup> o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">1 min</span>
 +
 +
</td>
 +
<td width="79" rowspan="3" >
 +
 +
<span style="font-family:&quot;">25 cycles</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">55 <sup>o</sup>C<sup></sup></span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">0.5 min</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">72 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">1.5 min<sup></sup></span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">72 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:&quot;">10 min</span>
 +
 +
</td>
 +
<td width="79" valign="top" >
 +
 +
<span style="font-family:&quot;">&nbsp;</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="58" valign="top" >
 +
 +
<span style="font-family:&quot;">4 <sup>o</sup>C</span>
 +
 +
</td>
 +
<td width="69" valign="top" >
 +
 +
<span style="font-family:宋体;">∝</span><span style="font-family:&quot;"></span>
 +
 +
</td>
 +
<td width="79" valign="top" >
 +
 +
<span style="font-family:&quot;">&nbsp;</span>
 +
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
    <div class="clear"></div> 
 +
<h4>3. Digestion</h4>
 +
<b>3.1 Digestion for confirmation</b>
 +
<p>Reaction System:</p>
 +
<table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" align="left" width="708" >
 +
<tbody>
 +
<tr>
 +
<td width="185" colspan="2" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<b><span style="font-size:10.0pt;font-family:&quot;">Single Digestion</span></b>
 +
</p>
 +
</td>
 +
<td width="183" colspan="2" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<b><span style="font-size:10.0pt;font-family:&quot;">Double Digestion</span></b>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="98" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">Plasmid</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">5</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">Plasmid</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">5</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="98" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Eco</span></i><span style="font-size:10.0pt;font-family:&quot;">R
 +
  I</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">1</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Eco</span></i><span style="font-size:10.0pt;font-family:&quot;">R
 +
  I</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">1</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="98" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">/</span></i><span style="font-size:10.0pt;font-family:&quot;"></span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">&nbsp;</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Pst</span></i><span style="font-size:10.0pt;font-family:&quot;"> I</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">1</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="98" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">10*H
 +
  buffer</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">1</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">10*M
 +
  buffer</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">1</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="98" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">ddH<sub>2</sub>O</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">3</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">ddH<sub>2</sub>O</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">2</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="98" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">Total
 +
  Volume</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">10</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">Total
 +
  Volume</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">10</span>
 +
</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="clear"></div>
 +
<p>Reaction condition: 37°C for 1 hours.</p>
 +
<div class="clear"></div>
 +
<b>3.2 Digestion for ligation</b>
 +
<p>Reaction System:</p>
 +
<table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" align="left" width="553" style="width:414.8pt;border:none;">
 +
<tbody>
 +
<tr>
 +
<td width="172" colspan="2" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<b><span style="font-size:10.0pt;font-family:&quot;">A + B</span></b>
 +
</p>
 +
</td>
 +
<td width="183" colspan="2" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<b><span style="font-size:10.0pt;font-family:&quot;">B + A</span></b>
 +
</p>
 +
</td>
 +
<td width="99" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<b><span style="font-size:10.0pt;font-family:&quot;">Volume (μl)</span></b>
 +
</p>
 +
</td>
 +
<td width="98" valign="top" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<b><span style="font-size:10.0pt;font-family:&quot;">Volume (μl)</span></b>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="85" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">A</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">B</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">A</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">B</span>
 +
</p>
 +
</td>
 +
<td width="99" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">30</span>
 +
</p>
 +
</td>
 +
<td width="98" valign="top" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">50</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="85" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Eco</span></i><span style="font-size:10.0pt;font-family:&quot;">R I</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">EcoR</span></i><span style="font-size:10.0pt;font-family:&quot;"> I</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Xba</span></i><span style="font-size:10.0pt;font-family:&quot;"> I</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Spe </span></i><span style="font-size:10.0pt;font-family:&quot;">I</span>
 +
</p>
 +
</td>
 +
<td width="99" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">3</span>
 +
</p>
 +
</td>
 +
<td width="98" valign="top" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">5</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="85" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Spe</span></i><span style="font-size:10.0pt;font-family:&quot;"> I</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Xba </span></i><span style="font-size:10.0pt;font-family:&quot;">I</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Pst</span></i><span style="font-size:10.0pt;font-family:&quot;"> I</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<i><span style="font-size:10.0pt;font-family:&quot;">Pst</span></i><span style="font-size:10.0pt;font-family:&quot;"> I</span>
 +
</p>
 +
</td>
 +
<td width="99" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">3</span>
 +
</p>
 +
</td>
 +
<td width="98" valign="top" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">5</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="85" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">10*H buffer</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">10*M buffer</span>
 +
</p>
 +
</td>
 +
<td width="87" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">10*M buffer</span>
 +
</p>
 +
</td>
 +
<td width="96" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">10*H buffer</span>
 +
</p>
 +
</td>
 +
<td width="99" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">4</span>
 +
</p>
 +
</td>
 +
<td width="98" valign="top" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">8</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="355" colspan="4" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">ddH<sub>2</sub>O</span>
 +
</p>
 +
</td>
 +
<td width="99" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">0</span>
 +
</p>
 +
</td>
 +
<td width="98" valign="top" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">12</span>
 +
</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="355" colspan="4" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<b><span style="font-size:10.0pt;font-family:&quot;">Total Volume</span></b>
 +
</p>
 +
</td>
 +
<td width="99" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">40</span>
 +
</p>
 +
</td>
 +
<td width="98" valign="top" >
 +
<p class="MsoListParagraph" align="center" style="text-align:center;text-indent:0cm;">
 +
<span style="font-size:10.0pt;font-family:&quot;">80</span>
 +
</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div class="clear"></div>
 +
<br/>
 +
<h4>4. Ligation</h4>
 +
<table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border:none;">
 +
<tbody>
 +
<tr>
 +
<td width="142" >
-
<div class="img-border">
+
<span style="font-size:12.0pt;font-family:&quot;">&nbsp;</span>
-
<img src="https://static.igem.org/mediawiki/2013/0/05/Xmusoftware_outreach_pho13.jpg" width =200px height=200px class="border alignleft" alt="" />
+
-
</div>
+
</td>
-
<p>
+
<td width="142" >
-
We held a garden party and related exhibition with the theme of synthetic biology and iGEM in the Gui Huashan Building, Xiamen University on July 17th afternoon.  The whole activity included three parts——synthetic biology exhibition, game and the Killers of Three Kingdoms cards presentation. To help the participants learn synthetic biology is a challenging but attractive process. These high school students could enjoy the interests in science and technology, know the knowledge about biology and think over ethics in life science through our activities. <br/> <br/>  
+
-
We ushered the students to our conference theatre, having them participate in any activities they are interested in and giving them attracting presents. We also gave every participant our badge.<br/> <br/>  
+
<b><span style="font-size:12.0pt;font-family:&quot;">Volume (μl)</span></b>
-
The synthetic biology exhibition was composed by some display boards including knowledge of synthetic biology, program display on iGEM, biological safety and genetic engineering. Some information and pictures were from iGEM wiki. We made this exhibition following the lecture to improve our HP effects. <br/> <br/>  
+
-
Garden party game was the most important parts in order to enhance the interest. It was composed with three parts—Riddles written on lanterns,Foldit computer games and finding the differences between DNA molecules. In the first game, we find some riddles related to gene and biology and wrote them on lanterns. If the participant could find the answer then he would win a prize. Foldit, a computer game liked by the high school students since they can learn from entertainment, folds proteins and tests their stabilities with four people competing in it.  Finding the differences between DNA molecules, is to find two or three differences between two analogous DNA pictures. From this game participants could be aware of DNA structure more deeply and practice their precision. We accumulated every students’ points and the final top 10 won the prize.<br/> <br/>  
+
</td>
-
Our special prize is the cards——Killers of Three Kingdoms, very popular in China. There are abundant role-relationship and mutual promotion and restraint between every cards, just like Bong in America!!!Our team designed this kind of cards particularly for the HP, combining synthetic biology with the cards. Some high school students even play the cards with our plain guidance, making us think it’s an interesting and enjoyable game. Participants hoped that we could  send to them more cards and give them designing pictures. From this game we had a better interaction.<br/> <br/>  
+
</tr>
-
Finally our team organized a special link for these high school students. We show them our culture strains so that they can get an access to germiculture.  <br/> <br/>  
+
<tr>
-
After the garden party as well as the exhibition, these senior high school students signed on our whiteboard and had a group photo taken with us at our request. We made informal interviews with some of them and get a satisfactory feedback. They wished we could hold the activities next year and they would try their best to popularize synthetic biology and iGEM. A student, from Shaowu No.1 Middle School Of Fujian, expressed his hope that we could come to their hometown to help people in the countryside to get a correct understanding of genetic engineering. As I know, some people even regard transgenosis as poisons. This is also our hope that more and more people can learn something about transgenic products via this competition and have a better understanding of genetic engineering. After all, we, as iGEMers, not only need to do experiments in laboratory, but also help synthetic biology understood by the public. <br/>
+
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">Insert</span>
 +
 +
</td>
 +
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">V<sub>1</sub></span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">Vector</span>
 +
 +
</td>
 +
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">V<sub>2</sub></span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">10*ligation
 +
  buffer</span>
 +
 +
</td>
 +
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">1</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">T4
 +
  Ligase</span>
 +
 +
</td>
 +
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">1</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="142" >
 +
 +
<b><span style="font-size:12.0pt;font-family:&quot;">Total Volume</span></b>
 +
 +
</td>
 +
<td width="142" >
 +
 +
<span style="font-size:12.0pt;font-family:&quot;">10</span>
 +
 +
</td>
 +
</tr>
-
</p>
+
</tbody>
-
<div class="clear"></div>
+
</table>
-
    <div class="clear"></div>  
+
 
 +
<div class="clear"></div>
 +
<br/>  
 +
<h4>5. Transformation</h4>
 +
<b> 5.1  Chemical Competent Cell Preparation (<i>E.coli</i>) </b><br/>
 +
<p class="MsoNormal" style="margin-left:7.1pt;">
 +
Activate the <i>E.coli </i>strain on LB-plate from glycerol stock under the condition of 37<sup>o</sup>C
 +
for 12 hours.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:7.1pt;">
 +
Pre-culture single-colony in 10 mL LB medium under
 +
the condition of 37<sup>o</sup>C, 200 rpm for 12 hours.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:7.1pt;">
 +
Add 200 μl pre-culture into 20 mL LB medium under
 +
the condition of 37<sup>o</sup>C, 200 rpm for 2 hours.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:7.1pt;">
 +
Place on ice for 30 min. Aliquot into sterile 1.5 mL
 +
tubes and spin down at 4000 rpm for 10 min at 4<sup> o</sup>C,
 +
then discard supernatant.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:7.1pt;">
 +
Gently
 +
re-suspend each pellet with 1 mL0.1 M ice cold CaCl<sub>2</sub>-MgCl<sub>2</sub><span class="apple-converted-space">&nbsp;solution</span>.
 +
Centrifuge 4000 rpm for 10 min and discard supernatant.<span class="apple-converted-space">&nbsp;</span><span class="apple-converted-space"></span>
 +
</p>
 +
<p class="MsoNormal" style="margin-left:7.1pt;">
 +
Re-suspend
 +
each pellet on ice in 200 μL 0.1 M ice cold CaCl<sub>2</sub>-MgCl<sub>2</sub><span class="apple-converted-space">&nbsp;solution</span><span class="apple-converted-space"></span>
 +
</p>
 +
 
 +
<b> 5.2 Chemical Competent Cell Preparation (<i>E.coli</i> with plasmid(s)) </b>
 +
<p>When you prepare the competent cell of <i>E.coli</i> with plasmid(s), you need add half-concentration of appropriate antibiotic(s) in LB.</p>
 +
 
 +
<b> 5.3 General Transformation </b>
 +
<p>
 +
Add 1 μL plasmid or 10 μL ligation system
 +
into 100 μL fresh chemical competent cells and mix gently. Leave on ice for 30
 +
minutes.
 +
</p>
 +
 +
Heat shock tubes in 42<sup>o</sup>C water
 +
bath for 90 seconds, then place them on ice for 10 minutes immediately.
 +
</p>
 +
<p class="MsoNormal" align="left" >
 +
Incubate for 1~2 hr with 200 rpm shaking at 37°C. When the antibiotic of vector is
 +
ampicillin, the time of incubate is 1 hr.
 +
</p>
 +
 +
<p>Add
 +
600 μL fresh LB medium into each tube.
 +
</p>
 +
<p>
 +
Spread 100
 +
μL on an LB solid medium plate containing the appropriate antibiotics and incubate overnight
 +
at 37°C
 +
</p>
 +
<b> Two or three plasmids Transformation </b>
 +
<p class="MsoNormal" style="margin-left:7.1pt;">
 +
Usually we just transform one plasmid into
 +
host cell, but sometimes we need transform two or three plasmids. Plasmid
 +
compatibility is considerable. Generally different replication origin means
 +
they are compatibility. We transform plasmids one by one from the low copy
 +
number to high copy number.
 +
</p>
 +
  <div class="clear"></div>
 +
 
</div><!-- .center-block-page -->
</div><!-- .center-block-page -->
Line 540: Line 1,426:
<!-- END WRAPPER -->    
<!-- END WRAPPER -->    
-
<!-- START WRAPPER SECTION -->
 
-
<div id="wrapper"  style="background:#ffcc66">
 
 +
<div id="wrapper2">
<style>
<style>
-
p {text-align:justify}
+
#wrapper2 {background:#ffe0e0}
</style>
</style>
-
        <div id="separator">
+
<div id="separator">
<div class="center-block">
<div class="center-block">
-
<a name="BioBang"></a>
+
<a name="SDS-PAGE"></a>
-
<h3>BioBang</h3>
+
<h3>2. SDS-PAGE</h3>
-
<span></span>
+
</div>
</div>
</div>
</div>
Line 556: Line 1,441:
<!-- START CONTENT -->
<!-- START CONTENT -->
<div class="center-block-page clearfix">
<div class="center-block-page clearfix">
-
+
<h4>Preparation</h4>
-
<p>
+
<b>2.1 Bacterial strains:</b>
-
It’s a great honor for me to introduce our attractive “byproduct”——Bio Bang. We designed these special card for these high school students who were the top ten on our scoring list. These sets of cards——we called them Bio Bang——were inspired by War Within Three Kingdoms, which are very popular in China,just like Bang! in America. It consists of abundant role-relationship and mutual promotion and restraint between every cards. The Bio Bang is themed with synthetic biology, including genetic engineering experiments, biological materials and equipment. We’ve also assimilate knowledge of biosafety into these set of cards in order to popularize knowledge of biosafety in lab, public and environment. This kind of propaganda tool is very useful to make different knowledge about genetic engineering and biology get across to high school students. Some example of them are shown as follows:</p>
+
<p>The experimental strains:<i>E.coli</i></p>
-
<img src="https://static.igem.org/mediawiki/2013/3/3c/Xmu-bbImage001.jpg" width =200px height=300px class="border alignleft" alt="" />
+
-
<img src="https://static.igem.org/mediawiki/2013/5/58/Xmu-bbImage003.jpg" width =200px height=300px class="border alignleft" alt="" />
+
-
    <div class="clear"></div>  
+
-
<img src="https://static.igem.org/mediawiki/2013/e/e7/Xmu-bbImage004.jpg" width =200px height=300px class="border alignleft" alt="" />
+
-
<img src="https://static.igem.org/mediawiki/2013/c/cc/Xmu-bbImage005.jpg" width =200px height=300px class="border alignleft" alt="" />
+
-
<img src="https://static.igem.org/mediawiki/2013/0/08/Xmu-bbImage009.jpg" width =200px height=300px class="border alignleft" alt="" />
+
-
    <div class="clear"></div>  
+
<b>2.2 The main instruments and reagents</b>
-
<p>
+
<br/>
 +
<i>Instruments</i>
 +
<p>autopipets, electrophoresis meter, Vortex oscillator, Labscan Scanner, decolorization table, desk centrifuge, temperature metal bath, electronic balance, acidometer, enzyme-labeled instrument, superclean bench, constant  temperature  vibrator,  shaking table.</p>
-
We’ve dreamed about perfecting the rules and designing and sold it out to raise money for our project. But it didn’t come true owing to our tight schedule. However, we decided to make a English version Bio Bang as presents for other iGEMers. Just wait for us coming!
+
<i>Reagents</i>
-
 
+
<p>LB liquid medium: yeast powder 5.0 g ;Tryptone 10.0 g ;sodium chloride
-
</p>
+
10.0 g;deionized water to produce 1000 ml medium.
-
+
</p>
 +
<p>LB solid medium: Put 1.5 g agar into every 100 ml liquid medium.</p>
 +
<i>Preparation of electrophoretic solution</i>
 +
<p class="MsoListParagraph">
 +
ddH<sub>2</sub>O;4 X Running Gel Buffer (PH 8.8,1.5 M Tris-HCl);4 X Running Gel Buffer (pH 6.8,1.5 M Tris-HCl);Monomer Solution;10% sodium dodecylsulphate(SDS);10% Ammonium Persulfate(APS);N,N,N',N'- tetramethylethylenediamine(TEMED);loading buffer;Tank Buffer;industrial alcohol; Marker ; dying
 +
liquor;destainer.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:20.25pt;">
 +
(1) Preparation of 4 X Running Gel
 +
Buffer (pH 8.8,1.5 M Tris-HCl): 36.3 g Tris-base is dissolved in 150 ml deionized water. Then use HCl
 +
solution to adjust the pH of solution to 8.8. At last use deionized water to produce 200 ml
 +
solution.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:20.25pt;">
 +
(2) Preparation of 4 X Running Gel
 +
Buffer (pH 6.8,1.5 M Tris-HCl): 3.0 g&nbsp;
 +
Tris-base is dissolved in 40 ml deionized water. Then use HCl solution to adjust the pH of
 +
solution to 6.8. At last use deionized
 +
water to produce 100 ml solution.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:20.25pt;">
 +
(3)Preparation
 +
of 10% SDS: 10g SDS is weighted before being dissolved . Use&nbsp; distilled water to produce 100ml solution.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:20.25pt;">
 +
(4)Preparation
 +
of 10% APS: 1 g APS is weighted before being dissolved. Use&nbsp; distilled water to produce 10ml solution.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:20.25pt;">
 +
(5)
 +
Preparation of 10X Tank Buffer: 30.28 g Tris-base, 144.13 g glycine and 10 g SDS
 +
is weighed before being dissolved. Use distilled water to produce 1000ml
 +
solution. Use the solution as-prepared 100 ml mixed with 900 ml distilled water
 +
to dilute 10 times.
 +
</p>
 +
<p class="MsoNormal" style="margin-left:20.25pt;">
 +
(6)
 +
Preparation of Kaumas Coomassie
 +
brilliant blue staining solution: 0.5 g Coomassie Brilliant blue R250 is
 +
weighted before being dissolved in 800 ml industrial alcohol. After dissolving,
 +
140 ml acetic acid is added and Use&nbsp;
 +
distilled water to produce 2000ml solution
 +
</p>
 +
<p class="MsoNormal" style="margin-left:20.25pt;">
 +
(7)
 +
Preparation of destainer: 200
 +
ml industrial alcohol is mixed with 50 ml acetic acid. Use distilled water to
 +
produce 1000ml solution.
 +
</p>
 +
<p class="MsoNormal" style="text-indent:20.0pt;">
 +
Preparation
 +
of 10% gel.
 +
</p>
 +
<div class="clear"></div>
<div class="clear"></div>
-
    <div class="clear"></div>  
+
<h4>2.2. Bacteria culture</h4>
-
+
<b>Strain activation</b>
-
</div><!-- .center-block-page -->
+
<p>Pick <i>Escherichia coli</i> colony from LB plate and subculture it into 20 ml conical beaker with 50 ml LB broth followed anaerobic culture 12 h under 37 °C . Then culture 1% concentration into conical beaker with 50 ml LB broth to subculture, which is the activated germ liquid.
 +
</p>
 +
<b>Strain culture</b>
 +
<p>Subculture 1% activated germ liquid into every conical beaker with LB broth. Then culture the microbe in the shaking table ,under 37 °C ,200 rpm.</p>
 +
<h4> 2.3 Protein electrophoresis</h4>
 +
<b>Preparation of Samples</b>
-
<!-- END CONTENT -->
+
-
</div><!-- #wrapper -->
+
<p class="MsoNormal" align="left" >
 +
- The samples to be tested are cultured in the
 +
Basal Medium with appropriate antibiotics, and take 200 μL bacteria liquid to
 +
determine its OD<sub>600</sub> at appropriate time.<br />
 +
- Dilute or concentrate the next 200 μL bacteria liquid in order to let the OD<sub>600</sub> equals to 4.0 while the computational formula is the actual OD<sub>600</sub> *
 +
200=2.0 * X, and X presents the total volume of the bacteria liquid after being
 +
diluted or concentrated while its unit is μL as well.<br />
 +
- Add 30 μL diluted or concentrated liquid into corresponding 1.5 mL
 +
centrifugal tubes, then mix up them with 10 μL loading buffer. <br />
 +
- Put these centrifugal tubes into metal bath and heat them in 100 °C  in around 5 to 8 min,
 +
then centrifuge them at the speed of 13000 rpm for 5 min, the supernatant is
 +
what we need.
 +
</p>
 +
<b>Manufacture Albumen Gel</b>
 +
<p>- Prepare a clear centrifuge tube in the capacity of 50 mL, and make running gel, high concentration one, following the formula below. Then mix up them and pour the mixture into a glass pane.</p>
 +
<p class="MsoNormal" style="margin-left:7.1pt;">
 +
<div align="center">
 +
<table class="MsoNormalTable ke-zeroborder" border="0" cellspacing="0" cellpadding="0" width=600>
 +
<tbody>
 +
<tr>
 +
<td width="444" colspan="2" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">Running Gel</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="444" colspan="2" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">Final Gel Concentration (5 mL; 1 ea ;1.0 mm
 +
  thick; 10%)</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">ddH<sub>2</sub>O</span>
 +
 +
</td>
 +
<td width="103" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">2 mL</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">4 X Running Gel Buffer (pH 8.8,1.5 M Tris-HCl)</span>
 +
 +
</td>
 +
<td width="103" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">1.25 mL</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">Monomer Solution</span>
 +
 +
</td>
 +
<td width="103" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">1.65 mL</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">10% SDS</span>
 +
 +
</td>
 +
<td width="103" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">50 uL</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">10% Ammonium Persulfate</span>
 +
 +
</td>
 +
<td width="103" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">50 uL</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">TEMED</span>
 +
 +
</td>
 +
<td width="103" valign="top" style="border:solid black 1.0pt">
 +
 +
<span style="font-size:10pt;font-family:'Times New Roman', serif;">5 uL</span>
 +
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</div>
 +
</p>
-
<!-- END WRAPPER -->
+
<p>- Add some absolute alcohol to planish the top of gel. There will be approximately 60 min for its solidification. </p>
-
<div id="wrapper3" style="background:#99ccff">
+
<p>- After solidification, pour out the alcohol and make stacking gel following the formula below. Then mix them up, add the solution onto the running gel in the glass panes until it being filled up with the gel. Insert a clean comb into stacking gel. Wait for about 40 min for stacking gel solidification.
-
<div id="separator">
+
</p>
-
       
+
<p class="MsoNormal" style="margin-left:7.1pt;">
-
<div class="center-block">
+
<div align="center">
-
<a name="Surveys"></a>
+
<table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border:none;" width=700>
-
<h3>Surveys</h3>
+
<tbody>
-
</div>
+
<tr>
 +
<td width="444" colspan="2" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">Running Gel</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="444" colspan="2" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">Final Gel
 +
  Concentration (5 ml; 1 ea ;1.0 mm thick; 10%)</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">ddH<sub>2</sub>O</span>
 +
 +
</td>
 +
<td width="103" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">2.7 ml</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">4
 +
  X Running Gel Buffer (PH 8.8,1.5 M Tris-HCl)</span>
 +
 +
</td>
 +
<td width="103" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">1.17 ml</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">Monomer
 +
  Solution</span>
 +
 +
</td>
 +
<td width="103" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">0.6 ml</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">10%
 +
  SDS</span>
 +
 +
</td>
 +
<td width="103" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">45 ul</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">10%
 +
  Ammonium Persulfate</span>
 +
 +
</td>
 +
<td width="103" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">45 ul</span>
 +
 +
</td>
 +
</tr>
 +
<tr>
 +
<td width="341" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">TEMED</span>
 +
 +
</td>
 +
<td width="103" valign="top" >
 +
 +
<span style="font-size:10.0pt;font-family:&quot;">5 ul</span>
 +
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
</div>
</div>
 +
</p>
 +
<b> Electrophoresis </b>
 +
<p class="MsoNormal" align="left" >
 +
- Take out the glass pane with finished gel and
 +
then fasten it in an electrophoresis tank. Add some 1×Tank Buffer to detect whether liquid leak or not.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br />
 +
- Take out the comb slowly and use pipette to add approximately 10 to 20 μL
 +
processed samples into the wells in stacking gel. <br />
 +
- Add 1×Tank Buffer until the
 +
liquid level is above the platinum line in the electrophoresis tank. <br />
 +
- Cover up the electrophoresis tank and connect it with the electrophoresis
 +
device. Set the program 80 V-30 min in one step and 120 V- 60 min in two step then start it up. <br />
 +
- When the blue marker band run to the bottom of running gel, stop the device.
 +
</p>
 +
<b>Dyeing (Colloidal Coomassie Brilliant Blue)</b>
-
<!-- START CONTENT -->
 
-
<div class="center-block-page clearfix">
 
-
 
-
 
-
<h4>Surveys on The Popularization of Synthetic Biology and iGEM
 
-
in high school students of Fujian Province<br/>
 
-
</h4>
 
<br/>
<br/>
-
<p>
+
<p class="MsoNormal" align="left" >
-
Our team conducted a synthetic biology survey before our lecture entitled "Contemporary introduction to synthetic biology". hWe wanted to compare how much they knew about synthetic biology and iGEM before the lecture with what their knowledge about them after our presentation in order to assess our work. We’ve prepared 300 questionnaires and handed out 227 of them. Among these drew-back questionnaires, 180 of them are valid. We put 12 questions on the questionnaire as below.<br/> <br/>
+
- Take out the gel and put it into a clean petri
-
1. Have you ever heard about synthetic biology before our lecture? <br/>
+
dish. And add appropriate Commassie Blue Staining Solution. Please make sure
-
2. If you have, in which way it was accessible to you?<br/>
+
that the solution can cover all the gel. <br />
-
3. Which parts do you think are the most intriguing about synthetic biology after our lecture?<br/>
+
- Put the petri dish onto the orbital shaker and dye for approximate 1 h.<br />
-
4. Have you ever heard about iGEM before our lecture? <br/>  
+
- Pour out the staining solution then add enough destaining solution. Destain
-
5. If you have, in which way it was accessible to you? <br/>  
+
about 30 min. <br />
-
6. Are you willing to participate in iGEM after our lecture? <br/>
+
- Renew the destaining solution for about 2 or 3 times until the blue
-
7. If your answer is no, why? <br/>  
+
background of gel being taken off. <br />
-
8. What’s your reason for participation, why? <br/>
+
- Pour out the destaining solution and add appropriate water to clear it.
-
9. If you participate in iGEM in which aspects do to want to get supports? <br/>  
+
</p>
-
10. How much time can you spend on iGEM?<br/>
+
-
11. If you participate, you and your family are willing to afford in which aspects? Some survey results are shown as below:<br/>
+
-
<img src="https://static.igem.org/mediawiki/2013/8/87/Xmu-Image001.png" width =300px height=200px class="border alignleft" alt="" />
+
-
<img src="https://static.igem.org/mediawiki/2013/8/89/Xmu-Image002.png" width =300px height=200px class="border alignleft" alt="" />
+
-
<img src="https://static.igem.org/mediawiki/2013/6/60/Xmu-Image003.png" width =300px height=200px class="border alignleft" alt="" />
+
-
<img src="https://static.igem.org/mediawiki/2013/0/05/Xmu-Image004.png" width =300px height=200px class="border alignleft" alt="" />
+
-
<img src="https://static.igem.org/mediawiki/2013/a/a2/Xmu-Image005.png" width =300px height=200px class="border alignleft" alt="" />
+
<br/>
<br/>
-
</p>
 
-
    <div class="clear"></div>
 
-
<p>
 
-
Discussion<br/>
 
-
Two major findings have been obtained from this analysis.<br/> <br/>
 
-
First of all, we drew a conclusion that almost everyone learned a lot from our lecture although they tended to be unfamiliar about the details of synthetic biology. Comparing the number of people who know about iGEM and the number of people who are willing to participate in iGEM we think the number of people who are interested in iGEM increases nearly 60%. Just look at the reasons why they don’t want to participate, most people chose no time and no supports. Only few people claimed that they were not interested in it and far less people said they didn’t like biology at all. Considering that our high school students are under so much pressure from National College Entrance Examination and some high school are too poor to afford technologies and apparatus, our results indicated a really good reality that almost everyone was willing to accept this new science and technology. <br/> <br/>
 
-
Second, as for how to popularize iGEM and synthetic biology, we summarize two points. <br/> <br/>
 
-
On the one hand, popularizing iGEM and synthetic biology via internet can reach a good effect because nearly half of these high school students said they heard iGEM and synthetic biology from the internet. We all think wiki is really a good platform to popularize synthetic biology and communicate with other iGEMers. What’s more, publishing more books relating to synthetic biology and iGEM is also a good conduit to let synthetic biology and iGEM known by other people, especially the young. <br/> <br/>
 
-
  On the other hand, teacher and parents seem to have difficulties in accepting a new technology in China or in the whole world so that students can’t learn some interesting and impressive new science and technologies from adults. It’s very common in China but we, as a new generation, should try our best to realize the rapid changing of our society.<br/> <br/>
 
-
Future plans<br/> <br/>
 
-
Finally we drew some conclusion to improve our survey next time. In order to obtain results that are more valid we should launch a more widely circulated survey. In addition, perhaps more copies can be distributed at random to the general public. On the other hand, it’s really important to make an effective and smart questionnaire to get a more cogent result. Maybe we can make two kinds of questionnaire to hand out before and after the lecture to compare to get a better conclusion. <br/> <br/>
 
-
   
+
<b> Scanning</b>
-
   
+
<p>Scan the processed gel and save the picture for analysis.</p>
-
</p>
+
 
-
<div class="clear"></div>
+
-
+
-
+
</div><!-- .center-block-page -->
</div><!-- .center-block-page -->
Line 641: Line 1,767:
</div><!-- #wrapper -->
</div><!-- #wrapper -->
-
<!-- END WRAPPER -->
+
<!-- END WRAPPER -->  
<!-- START WRAPPER SECTION -->
<!-- START WRAPPER SECTION -->
-
<div id="wrapper"  style="background:#ffccff">
+
<div id="wrapper"  style="background:#E1F3FC">
<style>
<style>
Line 650: Line 1,776:
         <div id="separator">
         <div id="separator">
<div class="center-block">
<div class="center-block">
-
<a name="Theme_Fresco"></a>
+
<a name="Microfluidic_Protocol"></a>
-
<h3>Theme Fresco</h3>
+
<h3>3. Microfluidic Protocol</h3>
-
<span></span>
+
</div>
</div>
</div>
</div>
Line 658: Line 1,783:
<!-- START CONTENT -->
<!-- START CONTENT -->
<div class="center-block-page clearfix">
<div class="center-block-page clearfix">
-
+
<b>Sterilization of microfluidic chips</b>
-
<p>
+
<b>Pre-procession of DH5α</b>
-
Besides those activities related to synthetic biology we’ve mentioned above, we assisted XMU_Software in finishing a theme fresco in Furong Tunnel. <br/><br/>
+
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
-
<img src="https://static.igem.org/mediawiki/2013/8/8d/Xmu-Tf1.jpg" width =300px height=200px class="border alignleft" alt="" />
+
1.
-
<br/><br/></p>
+
Briefly, 200 μl of an overnight culture was diluted
-
    <div class="clear"></div>  
+
in 20 ml of LB medium plus antibiotics the day of the experiment.
-
<p>
+
</p>
-
Furong Tunnel can be said to be the most attractive scenery of Xiamen University, visited by more than 30,000 people per day statistically. We made this work in consideration of its attractiveness, which can greatly help us to popularize not only synthetic biology and iGEM but also our team. In fact, many students witnessed the fresco, maybe called doodles more appropriately, made by iGEMers from Xiamen University. Some of them took photos and asked us about iGEM. It improved a good interaction between our team and these students and tourists to popularize iGEM competition and synthetic biology.
+
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:0cm;">
 +
(Cm, 10 μl; tet, 25 μl for two plasmids gfp+aiiA and three
 +
plasmids gfp+aiiA+ndh)
 +
</p>
 +
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
 +
2.
 +
When cells reached
 +
an optical density (OD<sub>600 nm</sub>) of 0.1 (for about 2 hr or
 +
longer), 12 ml cells were spun down and resuspended in 1.2 ml of fresh media and loaded
 +
into the device.
 +
</p>
-
</p>
+
<br/>
-
+
<b>Pre-procession of LB</b>
-
<div class="clear"></div>
+
<p>Filter 20 ml LB through 0.20 mm filter, and then add 0.075% Tween 20 (15 μl) to it. This step prevents the cells from sticking to chip walls without any noticeable harm to the cells. Depending on your experiment, make sure to add antibiotics to the media.
-
    <div class="clear"></div>   
+
So take the chip, your bacteria and your LB together to the florescent microscope.
-
+
</p>
-
</div><!-- .center-block-page -->
+
-
<!-- END CONTENT -->
+
<b>Wetting the chip & Capturing images</b>
-
</div><!-- #wrapper -->
+
-
<!-- END WRAPPER -->
+
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
-
   
+
1.
-
<div id="wrapper3" style="background:#98fb98">
+
Place the chip under microscope at<i> 4×magnification </i>and carefully
-
 
+
examine the chip for dirt and collapsed channels. If the chip looks good
-
<div id="separator">
+
proceed to wetting.
-
       
+
</p>
-
<div class="center-block">
+
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
-
<a name="Collaborations"></a>
+
2.
-
<h3>Collaborations</h3>
+
Wash syringe with 75% ethanol, MQ and
-
</div>
+
LB in proper sequence, and repeat each cleaning for three times
-
</div>
+
</p>
-
 
+
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
-
<!-- START CONTENT -->
+
3.
-
<div class="center-block-page clearfix">
+
Attach the plastic pipe about 15 to
-
<h4>First</h4>
+
20 cm in length to both syringes, and then fill one syringe with fresh LB,
-
<div class="img-border">
+
while the other one is filled with cells. Make sure there are <i>no bubbles in the syringe or the pipe</i>.
-
<img src="https://static.igem.org/mediawiki/2013/9/99/Xmu-Image06.jpg" width =250px height=200px class="border alignleft" alt="" />
+
Bubbles can be removed by flicking the syringe or the pipe with your finger.
-
</div>
+
</p>
-
<p>
+
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
-
We collaborated with Purdue iGEM team to create a definitive characterization standard for the parts registry. All the teams participated required to fill out a survey which about the important items in the registry page. Purdue iGEM team worked out a standard protocol which contains all of the details. The other teams need to complete it better or put forward some questions and problems. To make this work easy, we had a video meeting with the Purdue iGEM team and USP Brazil team though the GOOGLE HANGOUTS. It really helped us to exchange the opinions just like face to face.<br/><br/>  
+
4.
-
As we know, the members of this collaboration are currently over 50 teams so that it is the biggest collaboration in the iGEM history. We feel awesome about what we did!<br/>
+
Carefully insert the pipes into two
-
+
ports: media and cell, and set syringes steady on the pumps.
-
   
+
</p>
-
   
+
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
-
</p>
+
5.
-
<div class="clear"></div>
+
Set the flow rate of the left pump
 +
(LB) to >0.115 ml/hr to wet
 +
the chip until there is no bubble can be seen on the chip <i>(At
 +
20×magnification)</i>. Then slow the flow rate of this pump which should
 +
be much slower than that of the right one. Media removal from the surface of
 +
the chip at the cell port can be best accomplished using a kimwipe.
 +
</p>
 +
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
 +
6.
 +
Set the flow rate of the right pump
 +
(Cells) to 10~20 μl/hr to let cells fill in trapping region on the chip. When
 +
the cell density has reached a proper number, media flow should be 11.5~115
 +
μl/hr and the right pump should be stopped.
 +
</p>
 +
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
 +
7.
 +
Allow the cells to grow in the traps
 +
for 3-5 doublings, depending on the living condition and cell types this should
 +
take 1-2 h.
 +
</p>
 +
<p class="MsoBodyText" style="margin-left:18.0pt;text-align:justify;text-indent:-18.0pt;">
 +
8.
 +
Set up the microscope software for
 +
manual image acquisition. Turn on the laser power and adjust the light to <i>Blue</i>. Choose an appropriate field
 +
with suitable cell density without any dirt or other disturb and keep capture
 +
an image every 5 minutes.&nbsp;
 +
</p>
 +
    <div class="clear"></div>  
 +
<b> Before the image taking, make sure you focus the right layer. </p><p>
 +
When the timer is beeping, make sure you press the button twice, or you can use the time on computer or your cell phone to record the time.
 +
</b>
 +
    <div class="clear"></div>   
    <div class="clear"></div>   
</div><!-- .center-block-page -->
</div><!-- .center-block-page -->
-
<!-- START CONTENT -->
 
-
<div class="center-block-page clearfix">
 
-
<h4>Second</h4>
 
-
<div class="img-border">
 
-
<img src="https://static.igem.org/mediawiki/2013/b/b8/Xmu-cImage001.jpg" width =300px height=200px class="border alignleft" alt="" />
 
-
</div>
 
-
<p>On April 5th, 2013, Haoqian Zhang, advisors of Peking University iGEM team, a famous iGEMer who is keeping on develop synthetic biology and iGEM in China, come to XIAMEN University in purpose of strengthening collaborations our two universities. During his visiting, Mr. Zhang took part in discussions between our two teams, XMU_ Software and XMU-China2013 and gave some good advice focusing on the feasibility,security,advantages and disadvantages of ideas raised by our team members. His words about teamwork was very impressive and educational. Finally, Mr. Zhang simply showed some interesting projects in the past as well as encouraged us all to try our best to perform this year's iGEM projects, which greatly encouraged us.<br/><br/>   
 
-
</p>
 
-
<div class="clear"></div>
 
-
    <div class="clear"></div>   
 
-
 
-
</div><!-- .center-block-page -->
 
-
<!-- START CONTENT -->
 
-
<div class="center-block-page clearfix">
 
-
<h4>Third</h4>
 
-
<p>
 
-
From June 24th to June29th, We provided parts BBa_C0061 and BBa_I0462 to the Toulouse iGEM team due to the high transform difficulty they described using this year’s distribution. Toulouse could directly use them for their cloning ,while we got some valuable feedback about our parts.<br/><br/>
 
-
   
 
-
</p>
 
-
<div class="clear"></div>
 
-
    <div class="clear"></div>   
 
-
 
-
</div><!-- .center-block-page -->
 
-
<!-- START CONTENT -->
 
-
<div class="center-block-page clearfix">
 
-
<h4>Fourth</h4>
 
-
<p>
 
-
In the summer vacation, we drew a doodle about 2013 iGEM in Furong tunnel in the camp with the member of software team of our university. This tunnel is not only a main stem but also a famous scenic spots, so that more than 30,000 people per day will pass by and look our doodle. In addition, the doodle is a quite novel approach to introduce synthetic biology and we believe that no team did it before.<br/> <br/>
 
-
We also held a garden party with software team where introduced iGEM and synthetic biology to 200 students of senior high school from the whole Fujian Province. Go to page…to see more<br/> <br/>
 
-
In the wet lab, we helped them to structure their plasmids, such as teaching how to ligation and digestion.<br/><br/>
 
-
   
 
-
</p>
 
-
<div class="clear"></div>
 
-
    <div class="clear"></div>   
 
-
 
-
</div><!-- .center-block-page -->
 
-
<div class="center-block-page clearfix">
 
-
<h4>Fifth</h4>
 
-
<div class="img-border">
 
-
<img src="https://static.igem.org/mediawiki/2013/b/bd/Xmu-cImage004.jpg" width =400px height=300px class="border alignleft" alt="" />
 
-
</div>
 
-
<p>
 
-
We’ve also had a really good communication with the iGEM team of Peking University. From August 11th to August 19th ,one of our team members went to Peking University to get a deep cooperation with them. Firstly we introduced our own project in this year and exchanged the opinions .They gave us some good suggestions in experiments and models. What’s more, they gave us one BioBrick of sfGFP (BBa_...), which gave a great help to our structure. This sfGFP allowed our circuit to generated a brighter fluorescence and made easier to obverse in the microfluidics.<br/>
 
-
   
 
-
   
 
-
</p>
 
-
<div class="clear"></div>
 
-
    <div class="clear"></div>   
 
-
 
-
</div><!-- .center-block-page -->
 
-
<div class="center-block-page clearfix">
 
-
<h4>Sixth</h4>
 
-
<p>We had a short meeting with 4 members of NJU iGEM team visiting us on June24th, 2013. We welcome all other visitors as well. During the meeting, we introduced our project this year to these visitors and exchanged some opinions with them. It can improve the connection and collaboration between different iGEM team in China.<br/><br/> 
 
-
</p>
 
-
<div class="clear"></div>
 
-
    <img src="https://static.igem.org/mediawiki/2013/0/07/Xmusoftware_outreach_pho6.jpg" width =400px height=300px class="border alignleft" alt="" />
 
-
<img src="https://static.igem.org/mediawiki/2013/6/6d/Xmu-cImage003.jpg" width =400px height=300px class="border alignleft" alt="" />
 
-
 
 
-
    <div class="clear"></div>   
 
-
 
-
</div><!-- .center-block-page -->
 
<!-- END CONTENT -->
<!-- END CONTENT -->
</div><!-- #wrapper -->
</div><!-- #wrapper -->
Line 773: Line 1,876:
<!-- END WRAPPER -->
<!-- END WRAPPER -->
-
<div id="wrapper3">
 
-
<style>
 
-
#wrapper2 {background:#fccccc}
 
-
</style>
 
-
<div id="separator">
 
-
       
 
-
<div class="center-block">
 
-
<a name="Acknowledgement"></a>
 
-
<h3>Acknowledgement</h3>
 
-
</div>
 
-
</div>
 
-
 
-
<!-- START CONTENT -->
 
-
<div class="center-block-page clearfix">
 
-
 
-
<p>We would like to extend our sincere gratitude to all those who helped XMU-China2013 during this year. Without your help we wouldn’t have completed this project successfully. Some pieces of our appreciation are as follows.<br/><br/>
 
-
 
-
Team XMU-Software, the dry lab team of our university. Thank you for giving us so much help on wiki and modeling.<br/><br/>
 
-
 
-
Graduates in Lab 571,Lab 580 and Lab 117, College of Chemistry and Chemical Engineering , especially Chiming Ye, Tingting Wu, Yu Zhang, Nan Wang and Jing Guo. Without your experimental guidance we wouldn't have managed to finish the whole project.<br/><br/>
 
-
 
-
Prof. Hasty and his student Arthur Prindle, Department of Bioengineering, University of California, San Diego, La Jolla, California. Thank you for your patient and warm reply to our questions.<br/><br/>
 
-
 
-
Prof. Ningshao Xia and his graduates, School of Life Sciences, Xiamen University,  for providing us equipment and experimental guidance on electroporation.<br/><br/>
 
-
 
-
Prof. Shoufa Han, Department of Chemistry, Xiamen University, for providing us  fluorescence microplate reader and his students for experimental and equipment guidance.<br/><br/>
 
-
 
-
Institute of Urban Environment, Chinese Academy of Sciences for providing us MALDI-TOF-MS.<br/><br/>
 
-
  </p>
 
-
<div class="clear"></div>
 
-
<img src="https://static.igem.org/mediawiki/2013/a/ae/Xmuh-Image001.jpg" width =400px height=300px class="border alignleft" >
 
-
<img src="https://static.igem.org/mediawiki/2013/a/a1/Xmuh-Image002.jpg" width =300px height=300px class="border alignleft" >
 
-
    <div class="clear"></div>
 
-
<p>
 
-
Fujian Chemical Society and Prof. Yihui Chen. It's very kind of you to give us great support in Human Practice.<br/><br/>
 
-
 
-
College of Chemistry and Chemical Engineering. You've got our six.<br/><br/>
 
-
 
-
Xiamen University Academic Administration. Thank you for supporting us.<br/><br/>
 
-
 
-
High school students from all over Fujian Province taking part in our Human Practice. Without your great support we couldn't make a successful Human Practice.<br/><br/>
 
-
 
-
Peking iGEM 2013. Thanks for your sfGFP and some pieces of advice. If not we couldn't finish our work.<br/><br/>
 
-
 
-
Finally, again we’d like to express my heartfelt appreciation to all these kindness and enthusiasm. Thank you. Thanks for your self-giving helps.<br/><br/>
 
-
 
-
 
-
   
 
-
</p>
 
-
<div class="clear"></div>
 
-
    <div class="clear"></div>   
 
-
 
-
</div><!-- .center-block-page -->
 
-
 
-
<!-- END CONTENT -->
 
-
</div><!-- #wrapper -->
 
-
 
-
<!-- END WRAPPER -->
 
<!-- START FOOTER -->
<!-- START FOOTER -->
-
<div class="divider-100-2px"></div>
 
  <div class="clear"></div>   
  <div class="clear"></div>   
<div id="home_s4"><center>
<div id="home_s4"><center>
-
<img src="https://static.igem.org/mediawiki/2013/c/cb/Xmusoftware_sponsor1.png" width="120px" height="120px">
+
<img src="https://static.igem.org/mediawiki/2013/c/cb/Xmusoftware_sponsor1.png" width="80px" height="80px">
-
<img src="https://static.igem.org/mediawiki/2013/1/18/Xmusoftware_sponsor2.png" width="120px" height="120px">
+
<img src="https://static.igem.org/mediawiki/2013/1/18/Xmusoftware_sponsor2.png" width="80px" height="80px">
-
<img src="https://static.igem.org/mediawiki/2013/7/7f/Xmusoftware_sponsor3.png" width="120px" height="120px">
+
<img src="https://static.igem.org/mediawiki/2013/6/6f/Xmu-Bg_header_mwlogo_notag.jpg" height="60px">
-
<img src="https://static.igem.org/mediawiki/2013/6/6c/Xmusoftware_sponsor4.png" width="120px" height="120px">
+
<img src="https://static.igem.org/mediawiki/2013/e/ed/Xmu-evernote.jpg" height="60px">
-
<img src="https://static.igem.org/mediawiki/2013/6/66/Xmusoftware_sponsor5.png" width="120px" height="120px">
+
-
<img src="https://static.igem.org/mediawiki/2013/7/77/Xmusoftware_sponsor6.png" width="120px" height="120px">
+
</center></div>
</center></div>
<div id="footer-menu">
<div id="footer-menu">
Line 851: Line 1,893:
<li><a href="https://2013.igem.org/Team:XMU-China/Notebook">Notebook</a></li>
<li><a href="https://2013.igem.org/Team:XMU-China/Notebook">Notebook</a></li>
                 <li><a href="https://2013.igem.org/Team:XMU-China/Software">Software</a></li>
                 <li><a href="https://2013.igem.org/Team:XMU-China/Software">Software</a></li>
-
<li><a href="https://2013.igem.org/Team:XMU-China/Human Practice">Human Practice</a></li>
+
<li><a href="https://2013.igem.org/Team:XMU-China/Human Practice">Outreach</a></li>
<li><a href="https://2013.igem.org/Team:XMU-China/Safety">Safety</a></li>
<li><a href="https://2013.igem.org/Team:XMU-China/Safety">Safety</a></li>
</ul>
</ul>

Latest revision as of 18:00, 26 October 2013

LinkUp - Multipurpose HTML Template

Content

1 Circuit Construction

  • 1. Material
  • 2. PCR Polymerase Chain Reaction (PCR)
  • 3. Digestion
  • 4. Ligation
  • 5. Transformation
  • 2 SDS-PAGE

  • 1. Preparation
  • 2. Bacteria culture
  • 3. Protein electrophoresis
  • 3 Microfluidic Protocol

  • 1. Pre-procession of DH5α
  • 2. Pre-procession of LB
  • 3. Wetting the chip & Capturing images
  • 1. Circuit Construction

    Material


    1.1 Culture medium: LB (Luria-Bertani)

    LB medium: 5g Yeast Extract, 10g Tryptone and 10g NaCl, add ddH2O to 1L.
    LB solid medium: 5g Yeast Extract, 10g Tryptone, 10g NaCl and 15 g agar, add ddH2O to 1L.


    1.2 Antibiotic
    Antibiotic Stock conc. Final conc. Solvent Sterilization
    Ampicillin 100 mg/ml 50 ug/ml ddH2O filtration
    Chloramphenicol 25 mg/ml 25 ug/ml Absolute ethyl alcohol  
    Tetracycline 40 mg/ml 20 ug/ml 75% absolute ethyl alcohol  
    Kanamycin 25 mg/ml 25 ug/ml ddH2O filtration

    Stock in -20°C

    1.3 Plasmid (Biobrick)
    Part Backbone Type Location Size (bp)
    BBa_K546000 pSB1C3 Signaling 2013-P1-12D 1964 2070
    BBa_I763020 pSB1C3 Intermediate RBS-GFP-TT 2013-P3-11H 914 2070
    BBa_F2621 pSB1A2 Signaling 2013-P2-21F 1158 2079
    BBa_K546001 pSB1C3 Device 2013-P1-12F 2135 2070
    Ba_J04450 pSB4K5 Reporter 2013-P5-5G 1429 3409
    BBa_J04450 pSB3T5 Reporter 2013-P5-7C 1429 3241
    BBa_J04450 pSB3T5 Reporter 2013-P3-3H 1429 3241
    1.4 Strain
    Strain Application Source
    E. coli DH5α cloning Lab stock
    E. coli BL21(DE3)   Lab stock
    E. coli BL21   From Taiwan
    E. coli MG1655    
    1.5 Primer
    Part Backbone Type Location Size (bp)
    part backbone
    BBa_K546000 pSB1C3 Signaling 2013-P1-12D 1964 2070
    BBa_I763020 pSB1C3 Intermediate RBS-GFP-TT 2013-P3-11H 914 2070
    BBa_F2621 pSB1A2 Signaling 2013-P2-21F 1158 2079
    BBa_K546001 pSB1C3 Device 2013-P1-12F 2135 2070
    Ba_J04450 pSB4K5 Reporter 2013-P5-5G 1429 3409
    BBa_J04450 pSB3T5 Reporter 2013-P5-7C 1429 3241
          2013-P3-3H    

    2. PCR Polymerase Chain Reaction (PCR)


    2.1 General PCR

    Reaction system:

    Template 1 μl
    Forward Primer 2 μl
    Reverse Primer 2 μl
    dNTP Mixture 8 μl
    10*Ex Buffer 5 μl
    Ex Taq 0.5 μl
    ddH2O 31.5 μl
    Total Volume 50 μl

    Reaction condition:

    94 oC 4 min  
    94 oC 1 min 30 cycles
    55 oC 0.5 min
    72 oC 1.5 min
    72 oC 10 min  
    4 oC  
    2.2 Fusion PCR

    Every biobrick has four sites: EcoR I, Xba I, Spe I and Pst I. When two parts were ligated after digestion by Xba I and Spe I, an 8 bp-size fragment (TACTAGAG) will appear between two parts. If we want to add a fragment in the end of a CDS, for example a fast-degradation tag LVA, the additional 8 bp-size fragment will affect the expression of the CDS. Fusion PCR will resolve this problem. Firstly, two pair primers are designed: F1 and R1 for gene A, F2 and R2 for gene B. The 5'-end of R1 and F2 have about 21-bp homologous region. Secondly, gene A and gene B are amplified via PCR independently. Thirdly, the purified gene A and gene B are fused together via PCR with F1 and R2.


    2.3 Colony PCR

    Reaction system:

    Template colony
    Forward Primer 1 μl
    Reverse Primer 1 μl
    dNTP Mixture 4 μl
    10*PCR Buffer 2.5 μl
    rTaq 0.25 μl
    ddH2O 16.25 μl
    Total Volume 25 μl

    Reaction condition:

    94 oC 4 min  
    94 oC 1 min 25 cycles
    55 oC 0.5 min
    72 oC 1.5 min
    72 oC 10 min  
    4 oC  

    3. Digestion

    3.1 Digestion for confirmation

    Reaction System:

    Single Digestion

    Double Digestion

    Plasmid

    5

    Plasmid

    5

    EcoR I

    1

    EcoR I

    1

    /

     

    Pst I

    1

    10*H buffer

    1

    10*M buffer

    1

    ddH2O

    3

    ddH2O

    2

    Total Volume

    10

    Total Volume

    10

    Reaction condition: 37°C for 1 hours.

    3.2 Digestion for ligation

    Reaction System:

    A + B

    B + A

    Volume (μl)

    Volume (μl)

    A

    B

    A

    B

    30

    50

    EcoR I

    EcoR I

    Xba I

    Spe I

    3

    5

    Spe I

    Xba I

    Pst I

    Pst I

    3

    5

    10*H buffer

    10*M buffer

    10*M buffer

    10*H buffer

    4

    8

    ddH2O

    0

    12

    Total Volume

    40

    80


    4. Ligation

      Volume (μl)
    Insert V1
    Vector V2
    10*ligation buffer 1
    T4 Ligase 1
    Total Volume 10

    5. Transformation

    5.1 Chemical Competent Cell Preparation (E.coli)

    Activate the E.coli strain on LB-plate from glycerol stock under the condition of 37oC for 12 hours.

    Pre-culture single-colony in 10 mL LB medium under the condition of 37oC, 200 rpm for 12 hours.

    Add 200 μl pre-culture into 20 mL LB medium under the condition of 37oC, 200 rpm for 2 hours.

    Place on ice for 30 min. Aliquot into sterile 1.5 mL tubes and spin down at 4000 rpm for 10 min at 4 oC, then discard supernatant.

    Gently re-suspend each pellet with 1 mL0.1 M ice cold CaCl2-MgCl2 solution. Centrifuge 4000 rpm for 10 min and discard supernatant. 

    Re-suspend each pellet on ice in 200 μL 0.1 M ice cold CaCl2-MgCl2 solution

    5.2 Chemical Competent Cell Preparation (E.coli with plasmid(s))

    When you prepare the competent cell of E.coli with plasmid(s), you need add half-concentration of appropriate antibiotic(s) in LB.

    5.3 General Transformation

    Add 1 μL plasmid or 10 μL ligation system into 100 μL fresh chemical competent cells and mix gently. Leave on ice for 30 minutes.

    Heat shock tubes in 42oC water bath for 90 seconds, then place them on ice for 10 minutes immediately.

    Incubate for 1~2 hr with 200 rpm shaking at 37°C. When the antibiotic of vector is ampicillin, the time of incubate is 1 hr.

    Add 600 μL fresh LB medium into each tube.

    Spread 100 μL on an LB solid medium plate containing the appropriate antibiotics and incubate overnight at 37°C

    Two or three plasmids Transformation

    Usually we just transform one plasmid into host cell, but sometimes we need transform two or three plasmids. Plasmid compatibility is considerable. Generally different replication origin means they are compatibility. We transform plasmids one by one from the low copy number to high copy number.

    2. SDS-PAGE

    Preparation

    2.1 Bacterial strains:

    The experimental strains:E.coli

    2.2 The main instruments and reagents
    Instruments

    autopipets, electrophoresis meter, Vortex oscillator, Labscan Scanner, decolorization table, desk centrifuge, temperature metal bath, electronic balance, acidometer, enzyme-labeled instrument, superclean bench, constant temperature vibrator, shaking table.

    Reagents

    LB liquid medium: yeast powder 5.0 g ;Tryptone 10.0 g ;sodium chloride 10.0 g;deionized water to produce 1000 ml medium.

    LB solid medium: Put 1.5 g agar into every 100 ml liquid medium.

    Preparation of electrophoretic solution

    ddH2O;4 X Running Gel Buffer (PH 8.8,1.5 M Tris-HCl);4 X Running Gel Buffer (pH 6.8,1.5 M Tris-HCl);Monomer Solution;10% sodium dodecylsulphate(SDS);10% Ammonium Persulfate(APS);N,N,N',N'- tetramethylethylenediamine(TEMED);loading buffer;Tank Buffer;industrial alcohol; Marker ; dying liquor;destainer.

    (1) Preparation of 4 X Running Gel Buffer (pH 8.8,1.5 M Tris-HCl): 36.3 g Tris-base is dissolved in 150 ml deionized water. Then use HCl solution to adjust the pH of solution to 8.8. At last use deionized water to produce 200 ml solution.

    (2) Preparation of 4 X Running Gel Buffer (pH 6.8,1.5 M Tris-HCl): 3.0 g  Tris-base is dissolved in 40 ml deionized water. Then use HCl solution to adjust the pH of solution to 6.8. At last use deionized water to produce 100 ml solution.

    (3)Preparation of 10% SDS: 10g SDS is weighted before being dissolved . Use  distilled water to produce 100ml solution.

    (4)Preparation of 10% APS: 1 g APS is weighted before being dissolved. Use  distilled water to produce 10ml solution.

    (5) Preparation of 10X Tank Buffer: 30.28 g Tris-base, 144.13 g glycine and 10 g SDS is weighed before being dissolved. Use distilled water to produce 1000ml solution. Use the solution as-prepared 100 ml mixed with 900 ml distilled water to dilute 10 times.

    (6) Preparation of Kaumas Coomassie brilliant blue staining solution: 0.5 g Coomassie Brilliant blue R250 is weighted before being dissolved in 800 ml industrial alcohol. After dissolving, 140 ml acetic acid is added and Use  distilled water to produce 2000ml solution

    (7) Preparation of destainer: 200 ml industrial alcohol is mixed with 50 ml acetic acid. Use distilled water to produce 1000ml solution.

    Preparation of 10% gel.

    2.2. Bacteria culture

    Strain activation

    Pick Escherichia coli colony from LB plate and subculture it into 20 ml conical beaker with 50 ml LB broth followed anaerobic culture 12 h under 37 °C . Then culture 1% concentration into conical beaker with 50 ml LB broth to subculture, which is the activated germ liquid.

    Strain culture

    Subculture 1% activated germ liquid into every conical beaker with LB broth. Then culture the microbe in the shaking table ,under 37 °C ,200 rpm.

    2.3 Protein electrophoresis

    Preparation of Samples

    - The samples to be tested are cultured in the Basal Medium with appropriate antibiotics, and take 200 μL bacteria liquid to determine its OD600 at appropriate time.
    - Dilute or concentrate the next 200 μL bacteria liquid in order to let the OD600 equals to 4.0 while the computational formula is the actual OD600 * 200=2.0 * X, and X presents the total volume of the bacteria liquid after being diluted or concentrated while its unit is μL as well.
    - Add 30 μL diluted or concentrated liquid into corresponding 1.5 mL centrifugal tubes, then mix up them with 10 μL loading buffer.
    - Put these centrifugal tubes into metal bath and heat them in 100 °C in around 5 to 8 min, then centrifuge them at the speed of 13000 rpm for 5 min, the supernatant is what we need.

    Manufacture Albumen Gel

    - Prepare a clear centrifuge tube in the capacity of 50 mL, and make running gel, high concentration one, following the formula below. Then mix up them and pour the mixture into a glass pane.

    Running Gel
    Final Gel Concentration (5 mL; 1 ea ;1.0 mm thick; 10%)
    ddH2O 2 mL
    4 X Running Gel Buffer (pH 8.8,1.5 M Tris-HCl) 1.25 mL
    Monomer Solution 1.65 mL
    10% SDS 50 uL
    10% Ammonium Persulfate 50 uL
    TEMED 5 uL

    - Add some absolute alcohol to planish the top of gel. There will be approximately 60 min for its solidification.

    - After solidification, pour out the alcohol and make stacking gel following the formula below. Then mix them up, add the solution onto the running gel in the glass panes until it being filled up with the gel. Insert a clean comb into stacking gel. Wait for about 40 min for stacking gel solidification.

    Running Gel
    Final Gel Concentration (5 ml; 1 ea ;1.0 mm thick; 10%)
    ddH2O 2.7 ml
    4 X Running Gel Buffer (PH 8.8,1.5 M Tris-HCl) 1.17 ml
    Monomer Solution 0.6 ml
    10% SDS 45 ul
    10% Ammonium Persulfate 45 ul
    TEMED 5 ul

    Electrophoresis

    - Take out the glass pane with finished gel and then fasten it in an electrophoresis tank. Add some 1×Tank Buffer to detect whether liquid leak or not.     
    - Take out the comb slowly and use pipette to add approximately 10 to 20 μL processed samples into the wells in stacking gel.
    - Add 1×Tank Buffer until the liquid level is above the platinum line in the electrophoresis tank.
    - Cover up the electrophoresis tank and connect it with the electrophoresis device. Set the program 80 V-30 min in one step and 120 V- 60 min in two step then start it up.
    - When the blue marker band run to the bottom of running gel, stop the device.

    Dyeing (Colloidal Coomassie Brilliant Blue)

    - Take out the gel and put it into a clean petri dish. And add appropriate Commassie Blue Staining Solution. Please make sure that the solution can cover all the gel.
    - Put the petri dish onto the orbital shaker and dye for approximate 1 h.
    - Pour out the staining solution then add enough destaining solution. Destain about 30 min.
    - Renew the destaining solution for about 2 or 3 times until the blue background of gel being taken off.
    - Pour out the destaining solution and add appropriate water to clear it.


    Scanning

    Scan the processed gel and save the picture for analysis.

    3. Microfluidic Protocol

    Sterilization of microfluidic chips Pre-procession of DH5α

    1. Briefly, 200 μl of an overnight culture was diluted in 20 ml of LB medium plus antibiotics the day of the experiment.

    (Cm, 10 μl; tet, 25 μl for two plasmids gfp+aiiA and three plasmids gfp+aiiA+ndh)

    2. When cells reached an optical density (OD600 nm) of 0.1 (for about 2 hr or longer), 12 ml cells were spun down and resuspended in 1.2 ml of fresh media and loaded into the device.


    Pre-procession of LB

    Filter 20 ml LB through 0.20 mm filter, and then add 0.075% Tween 20 (15 μl) to it. This step prevents the cells from sticking to chip walls without any noticeable harm to the cells. Depending on your experiment, make sure to add antibiotics to the media. So take the chip, your bacteria and your LB together to the florescent microscope.

    Wetting the chip & Capturing images

    1. Place the chip under microscope at 4×magnification and carefully examine the chip for dirt and collapsed channels. If the chip looks good proceed to wetting.

    2. Wash syringe with 75% ethanol, MQ and LB in proper sequence, and repeat each cleaning for three times

    3. Attach the plastic pipe about 15 to 20 cm in length to both syringes, and then fill one syringe with fresh LB, while the other one is filled with cells. Make sure there are no bubbles in the syringe or the pipe. Bubbles can be removed by flicking the syringe or the pipe with your finger.

    4. Carefully insert the pipes into two ports: media and cell, and set syringes steady on the pumps.

    5. Set the flow rate of the left pump (LB) to >0.115 ml/hr to wet the chip until there is no bubble can be seen on the chip (At 20×magnification). Then slow the flow rate of this pump which should be much slower than that of the right one. Media removal from the surface of the chip at the cell port can be best accomplished using a kimwipe.

    6. Set the flow rate of the right pump (Cells) to 10~20 μl/hr to let cells fill in trapping region on the chip. When the cell density has reached a proper number, media flow should be 11.5~115 μl/hr and the right pump should be stopped.

    7. Allow the cells to grow in the traps for 3-5 doublings, depending on the living condition and cell types this should take 1-2 h.

    8. Set up the microscope software for manual image acquisition. Turn on the laser power and adjust the light to Blue. Choose an appropriate field with suitable cell density without any dirt or other disturb and keep capture an image every 5 minutes. 

    Before the image taking, make sure you focus the right layer.

    When the timer is beeping, make sure you press the button twice, or you can use the time on computer or your cell phone to record the time.