Team:Hong Kong HKUST/wetlab/notebook

From 2014.igem.org

(Difference between revisions)
 
(70 intermediate revisions not shown)
Line 1: Line 1:
 +
{{Team:Hong_Kong_HKUST/shell |
<html>
<html>
<head>
<head>
-
<link rel="stylesheet" href="https://2014.igem.org/wiki/index.php?title=Template:Team:Hong_Kong_HKUST/anti-main.css&action=raw&ctype=text/css" type="text/css" >
+
-
<link rel="stylesheet" href="https://2014.igem.org/wiki/index.php?title=Template:Team:Hong_Kong_HKUST/indexpage.css&action=raw&ctype=text/css" type="text/css" >
+
-
<script src="http://ajax.aspnetcdn.com/ajax/jQuery/jquery-1.11.1.min.js"></script>
+
-
<script src="https://2014.igem.org/wiki/index.php?title=Template:Team:Hong_Kong_HKUST/access-menu.js&action=raw&ctype=text/css"></script>
+
</head>
</head>
 +
</html>|
 +
<html>
<body>
<body>
-
<nav role="navigation">
+
-
<ul class="access-menu">
+
-
<li><a href="#">Home</a></li>
+
-
<li>
+
-
<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor">Pneumosensor</a>
+
-
<ul class="access-submenu">
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_one">Sensing</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/module_two">Expressing</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/parts">Parts</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/data">Data</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization">Characterization</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/results">Results</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/future_work">Future Work</a></li>
+
-
</ul>
+
-
</li>
+
-
<li>
+
-
<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator">Riboregulator</a>
+
-
<ul class="access-submenu">
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/CR_TA_Feature_Page">Feature Page</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/RNA_devices_catalog">Catalog Page</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/parts">Parts</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/data">Data</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization">Characterization</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/results">Results</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/future_work">Future Work</a></li>
+
-
</ul>
+
-
</li>
+
-
<li>
+
-
<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice">Human Practice</a>
+
-
<ul class="access-submenu">
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit">Start-up Kit</a></li>
+
-
<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/handbook" >--Handbook</a></li>
+
-
<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/report" >--Report</a></li>
+
-
<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/database" >--Database</a></li>
+
-
<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/start-up_kit/interview" >--Interview</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach">Outreach</a></li>
+
-
<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/Workshop" >--Workshop</a></li>
+
-
<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/talks" >--Talk</a></li>
+
-
<li class= "indent_list"><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/outreach/isf_academy" >--ISF Academy</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/human_practice/safety_and_ethics">Safety and Ethics</a></li>
+
-
</ul>
+
-
</li>
+
-
<li>
+
-
<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team">Team</a>
+
-
<ul class="access-submenu">
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/members">Members</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/advisers">Advisers</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/instructors">Instructors</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/attribution">Attributions</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/team/acknowledgement">Acknowledgement</a></li>
+
-
<li><a href="https://igem.org/Team.cgi?year=2014">Official Team Profile</a></li>
+
-
</ul>
+
-
</li>
+
-
<li>
+
-
<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab">Wetlab</a>
+
-
<ul class="access-submenu">
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/notebook">Notebook</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/safety">Safety</a></li>
+
-
</ul>
+
-
</li>
+
-
<li>
+
-
<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/Achievements">Achievements</a>
+
-
<ul class="access-submenu">
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/acheivement/medal_requirement">Medal Requirements</a></li>
+
-
<li><a href="https://2014.igem.org/Team:Hong_Kong_HKUST/acheivement/deliverable">Deliverable</a></li>
+
-
</ul>
+
-
</li>
+
-
<li class="access_logo">
+
-
<a href="https://2014.igem.org/Main_Page"><img src= "https://static.igem.org/mediawiki/2014/5/55/Hkust_logo.gif"></a>
+
-
</li>
+
-
<li class="access_logo">
+
-
<a href="http://www.ust.hk/"><img src= "https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png"></a>
+
-
</li>
+
-
+
-
 
+
-
</ul>
+
-
</nav>
+
-
<!-- ================ do not touch any thing above this, dont even think about it =========================-->
+
-
+
-
+
<div id="content_container">
<div id="content_container">
-
<div class= "banner_area">
 
-
<img src= 'https://static.igem.org/mediawiki/2014/archive/3/3c/20140930022303!HKUST_2014_pneumosensor_banner.jpg' />
 
-
<!--banner should be larger and height should be more than 400px-->
 
-
</div>
 
-
<hr>
 
<div class='content_1'>
<div class='content_1'>
-
<h3>Notebook</h3>
+
<h3 style="text-align:center">Notebook</h3>
<table class="content_table" align= "center" valign= "top">
<table class="content_table" align= "center" valign= "top">
<tr class= "content_row">
<tr class= "content_row">
Line 105: Line 22:
<p>
<p>
Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and
Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and
-
research findings. The notebook for Pneuumonsensor and Riboregulator are displayed separately.  
+
research findings. The notebook for Project Pneuumonsensor and Project Riboregulator are displayed separately.  
-
+
</p>
</p>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
 +
<h3>Pneumosensor</h3>
   <script src="http://ajax.googleapis.com/ajax/libs/jquery/1.7.1/jquery.min.js"></script>
   <script src="http://ajax.googleapis.com/ajax/libs/jquery/1.7.1/jquery.min.js"></script>
   <script>
   <script>
Line 127: Line 50:
     .head {  
     .head {  
display:block;
display:block;
-
background:#FF9900;
+
background:#FFCA75;
border-radius:2px;
border-radius:2px;
margin-bottom:5px;
margin-bottom:5px;
Line 137: Line 60:
font-size: 35px;
font-size: 35px;
text-align: left;
text-align: left;
 +
color:black;
}
}
Line 169: Line 93:
<!-- JJJJJJJJJJJJ   UUUUUUUUUUUU N       N EEEEEEEEEEE   2 0 1 3---------------->
<!-- JJJJJJJJJJJJ   UUUUUUUUUUUU N       N EEEEEEEEEEE   2 0 1 3---------------->
-
<div id="satu" align="center"> <h2>June 2013</h2>
+
<div id="satu" align="center"> <h2>June 2014</h2>
    
    
       <div>
       <div>
       <a href="#" class="head">Week 1</a>
       <a href="#" class="head">Week 1</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>June xx</b> <br>
+
<br><b>June 9</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Transformation of pSB1C3-BBa_E0240, pSB1C3-BBa_B0032 and pSB1C3-BBa_K20600<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Transformation of BBa_B0034 and BBa_E0040 into DH10B<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
 
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
<br><b>June 10</b> <br>
-
<br><b>June xx</b> <br>
+
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Found reference promoter J23101 in BBa_I20260 from part registry<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Bacterial inoculation of BBa_B0034 and BBa_E0040<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
 
-
<br><b>June xx</b> <br>
+
<br><b>June 11</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Studied the Characteristic, function and mechnism of how promoters work<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Miniprep of BBa_B0034 and BBa_E0040<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
 
-
<br><b>June xx</b> <br>
+
<br><b>June 12</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      research on gene sequence of PcomX, how to quantify p~<i>comE</i>, range of concentration of  p~<i>comE</i> to use for characterization<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      explain of scar<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
 +
<br><b>June 13</b> <br>
 +
Wet lab<br>
 +
·      Looked for P<sub>combox</sub> gene sequence from the genomic DNA<br>
 +
·      research on gene sequence of PcomX, how to quantify p~<i>comE</i>, range of concentration of  p~<i>comE</i> to use for characterization<br>
       </div>
       </div>
Line 205: Line 131:
       <a href="#" class="head">Week 2</a>
       <a href="#" class="head">Week 2</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>June xx</b> <br>
+
 
 +
<br><b>June 16</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for P<sub>combox</sub> gene sequence<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Found a possible P<sub>combox</sub><br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Transformation of BBa_J04450 and BBa_I20260 into DH10B<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Bacterial inoculation of BBa_E0240<br>
-
<br><b>June xx</b> <br>
+
·   <br>
 +
<br><b>June 17</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for P<sub>combox</sub> gene sequence<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Found a possible P<sub>combox</sub><br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Searched for other possible P<sub>combox</sub> sequences<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Miniprep of BBa_E0240<br>
-
<br><b>June xx</b> <br>
+
·      Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br>
 +
·   <br>
 +
<br><b>June 18</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Designed primers for the P<sub>combox</sub> sequences<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Received a P<sub>combox</sub> sequence from Wellcome Trust Sanger Institute<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Miniprep of BBa_J04450(CHL) and BBa_I20260<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260<br>
-
<br><b>June xx</b> <br>
+
·      Digestion of BBa_J04450(CHL) and BBa_I20260<br>
-
Wet lab<br>
+
·      Gel electrophoresis of digested products<br>
-
·     Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      <br>
-
·     Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
<br><b>June 19</b> <br>
-
·     PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
· Digestion of pSB1C3-BBa_E0240 at EcoRI and SpeI<br>
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
· Miniprep of BBa_J04450(CHL) and BBa_I20260<br>
 +
· Digestion of BBa_J04450(CHL)<br>
 +
· Gel electrophoresis of digested product<br>
 +
· Bacterial inoculation of BBa_J04450(CHL)<br>
 +
· <br>
 +
<br><b>June 20</b> <br>
 +
· Amended primers of P<sub>combox</sub> sequences<br>
 +
· Miniprep of BBa_J04450(CHL)<br>
 +
· Digestion of BBa_J04450(CHL)<br>
 +
· Gel electrophoresis of digested product<br>
 +
· Designed primers for cloning <i>comD</i> and <i><i>comE</i></i><br>
 +
 
       </div>
       </div>
Line 235: Line 176:
       <a href="#" class="head">Week 3</a>
       <a href="#" class="head">Week 3</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>June xx</b> <br>
+
<br><b>June 23</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Transformation of BBa_B0015 from iGEM Kit Plate 2011 and BBa_K880005<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Primer design for PCR of comX-myc and <i>comW</i>-FLAG from genomic DNA of S. Pneumoniae NCTC 7465 strain<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Designed plasmid construct for P<sub>combox</sub><br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Bacterial inoculation of BBa_J04450(CHL)<br>
-
<br><b>June xx</b> <br>
+
-
Wet lab<br>
+
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
-
<br><b>June xx</b> <br>
+
-
Wet lab<br>
+
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
-
<br><b>June xx</b> <br>
+
-
Wet lab<br>
+
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
-
 
+
·   <br>
-
      </div>
+
<br><b>June 24</b> <br>
-
    </div>
+
-
<div>
+
-
      <a href="#" class="head">Week 4</a>
+
-
      <div class="content" align="left" style="display: none;">
+
-
<br><b>June xx</b> <br>
+
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Inoculation of BBa_B0015<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Designed new reverse primer for 100bp, 150bp, 160bp, 180bp and 249bp P<sub>combox</sub><br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Designed forward and reverse oligos of 67bp P<sub>combox</sub><br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Transformation of pSB1C3-BBa_I13504<br>
-
<br><b>June xx</b> <br>
+
·      Miniprep of BBa_J04450(CHL)<br>
 +
·      Transformation of BBa_J04450(KAN)<br>
 +
·      Bacterial inoculation of BBa_E0240<br>
 +
·   <br>
 +
<br><b>June 25</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Inoculation of BBa_K880005<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Inoculation of pSB1C3-BBa_E0240<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Miniprep of BBa_E0240<br>
-
<br><b>June xx</b> <br>
+
·      Digestion of BBa_E0240<br>
 +
·      Transformation of BBa_J04450(KAN)<br>
 +
·      Bacterial inoculation of BBa_J04450(CHL)<br>
 +
·      <br>
 +
·   <br>
 +
<br><b>June 26</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion of BBa_K880005 plasmid extraction product with SpeI and PstI, gel extraction and gel purification<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Gel electrophoresis of digested BBa_E0240<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Gel extraction and purification of digested BBa_E0240 to obtain the insert<br>
-
<br><b>June xx</b> <br>
+
·      Miniprep of BBa_J04450(CHL)<br>
 +
·      Digestion of BBa_J04450(CHL)<br>
 +
·      Gel electrophoresis of digested BBa_J04450(CHL)<br>
 +
·      Gel extraction and purification of digested BBa_J04450(CHL) to obtain pSB1C3<br>
 +
·      Bacterial inoculation of BBa_K880005<br>
 +
·   <br>
 +
<br><b>June 27</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Ligation of BBa_B0015 insert cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI as negative control<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Gel purification of pSB1C3-BBa_E0240<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Miniprep of BBa_K880005<br>
 +
·   <br>
Line 295: Line 229:
     </div>
     </div>
<div>
<div>
-
       <a href="#" class="head">Week 5</a>
+
       <a href="#" class="head">Week 4</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>June xx</b> <br>
+
<br><b>June 30</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Gel check of purified BBa_B0015 insert<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Transformation of ligated pSB1C3-K880005.B0015<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·   <br>
-
<br><b>June xx</b> <br>
+
 
-
Wet lab<br>
+
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
-
<br><b>June xx</b> <br>
+
-
Wet lab<br>
+
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
-
<br><b>June xx</b> <br>
+
-
Wet lab<br>
+
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
       </div>
       </div>
Line 341: Line 258:
-
<div id="satu" align="center"> <h2><center>July 2013</center></h2>
+
<div id="satu" align="center"> <h2><center>July 2014</center></h2>
    
    
   <div>
   <div>
       <a href="#" class="head">Week 1</a>
       <a href="#" class="head">Week 1</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br> <b> July 2 </b> <br>
+
<br> <b> July 2</b> <br>
-
Wet lab <br>
+
Wet lab<br>
-
·      Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter <br>
+
·      Arrival of primers order for comX-myc and <i>comW</i>-FLAG<br>
-
·      LB-Chloramphenicol plates (the previous ones were found contaminated) <br>
+
·      Dilution of primers and design of experiment for both comX-myc and <i>comW</i>-FLAG<br>
-
·      Inoculation of pEGFP-N1 for plasmid extraction <br>
+
·      Inoculation of ligated pSB1C3-K880005.B0015<br>
-
·      Transformation of P<i>fadBA</i> due chloramphenicol plates contamination <br>
+
·      Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240<br>
 +
·      Oligo annealing for PcomX<br>
 +
·      Bacterial inoculation of BBa_J04450 (KAN) (6 tubes), BBa_E0240 (CAM) (6 tubes), BBa_B0015 (KAN) (3 tubes)<br>
 +
·      PCR for cloning <i>comD</i> and <i><i>comE</i></i><br>
-
<br> <b> July 3 </b> <br>
 
-
·      Extraction of pEGFP-N1 plasmids by miniprep <br>
 
-
·      PCR for PPAR-alpha promoter amplification <br>
 
-
·      Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter <br>
 
-
<br> <b> July 4</b> <br>
+
<br> <b> July 3</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Inoculation of BBa_K817002  P<i>fadBA</i><br>
+
·      PCR of comX-myc using Taq DNA Polymerase with ThermoPol Buffer to check whether primers are functional<br>
-
·      PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
+
·      Gel check for comX-myc PCR product<br>
-
·      Ran gel for digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; Xba1 and BamH1 for FABP1 promoter; Ase1 and Xhol1 for GRP78<br>
+
·      PCR of comX-myc using Phusion High-Fidelity PCR Master Mix with HF Buffer<br>
 +
·      Gel check for comX-myc PCR product<br>
 +
·      Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 with XbaI and PstI<br>
 +
·      Received and stored the new common reverse primers for P<sub>combox</sub> and sense oligo and anti-sense oligo of 67bp P<sub>combox</sub><br>
 +
·      Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Gel purification of digested BBa_E0240 and BBa_J04450<br>
 +
·   Inoculation of pSB3K3-BBa_J04450<br>
 +
·      PcomX Transformation<br>
 +
·      PcomCDE Oligo annealing, ligation, transformation<br>
 +
·      J04450 Plasmid Extraction<br>
 +
·      E0240:Plasmid extraction ; Digestion; Gel electrophoresis; Gel extraction ; Gel purification<br>
 +
·      B0015: Plasmid extraction ; Nanodrop<br>
 +
·      <i>comE<sup>D58E</sup></i>: PCR<br>
 +
·        <br>
-
<br> <b> July 5</b> <br>
+
<br> <b> July 4</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Extraction of BBa_K817002 (P<i>fadBA</i>) <br>
+
·      PCR of comX-myc using Vent DNA Polymerase, PCR clean up<br>
-
·      Inoculation of pEGFP-N1<br>
+
·      Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI<br>
-
·      PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
+
·      <i>comE<sup>D58E</sup></i> :Phusion PCR<br>
-
·      Gel check, 0.8% gel for previously gDNA extraction<br>
+
·      E0240: Gel purification<br>
-
·      Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter<br>
+
·   <br>
-
·      Poured new LB-Kanamycin plates<br>
+
-
·      Transformation of <a href="http://parts.igem.org/Part:BBa_J176171">BBa_J176171</a><br>
+
-
Dry lab<br>
+
-
·      Discussion about re-design constructions, possible change for BBa_J176171 as mammalian expression vector<br>
+
-
·     Primer redesign for PPAR-alpha promoter and FABP1 promoter<br><br>
+
-
 
+
       </div>
       </div>
Line 384: Line 307:
       <a href="#" class="head">Week 2</a>
       <a href="#" class="head">Week 2</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
 +
<br> <b> July 7</b> <br>
 +
Wet lab<br>
 +
·      Digestion of comX-myc with XbaI and PstI<br>
 +
·      Gel check of digested comX-myc, digestion clean up<br>
 +
·      PCR of <i>comW</i>-FLAG using Vent DNA Polymerase<br>
 +
·      Gel check for <i>comW</i>-FLAG PCR product, PCR clean up<br>
 +
·      Dephosporylation of digested pSB1C3-K880005 backbone cut with SpeI and PstI<br>
 +
·      Gel purification of digested BBa_E0240 and BBa_J04450<br>
 +
·      Submitted primers for Gibson assembly of pSB3K3-100bpP<sub>combox</sub><br>
 +
·      Inoculation of pSB3K3-BBa_J04450<br>
 +
·      E0240 - Bacterial inoculation<br>
 +
·      pSB1C3-PcomX: Bacterial inoculation<br>
 +
·      pSB1C3- PcomCDE: Bacterial inoculation, Streaking<br>
 +
·      <i>comE<sup>D58E</sup></i> :PCR cleanup; Digestion; Ligation with pSB1C3; Transformation<br>
 +
·   <br>
 +
<br> <b> July 8</b> <br>
<br> <b> July 8</b> <br>
Wet lab<br>
Wet lab<br>
-
·      gDNA extraction from HepG2 cells<br>
+
·      Ligation of comX-myc cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI<br>
-
·      Restriction of BBa_K817002 P<i>fadBA</i><br>
+
·      2nd attempt of ligation of BBa_B0015 insert cut with XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut with SpeI and PstI as negative control<br>
-
·      Gel check for gDNA extraction<br>
+
·      Transformation of ligation products<br>
-
·      Inoculation of BBa_J176171, BBa_K817002 (P<i>fadBA</i>), pEGFP-N1 for plasmid extraction<br>
+
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
-
·      New primers for PPAR-alpha promoter and FABP1 promoter arrival<br>
+
·      Gel purification of digested BBa_J04450<br>
-
·      Ran gel for BBa_K817002 P<i>fadBA</i> restriction check, gel extraction and purification<br>
+
·      Received pCEPcin from Universite de Toulouse, France<br>
 +
·      J04450 (KAN): Digestion; Gel electrophoresis; Gel extraction<br>
 +
·      E0240: Plasmid extraction<br>
 +
·      pSB1C3-PcomX: Plasmid extraction<br>
 +
·      pSB1C3- PcomCDE: Plasmid extraction<br>
 +
·      <i>comE<sup>D58E</sup></i>: Bacterial inoculation; Ligation; Transformation<br>
 +
·   <br>
<br> <b> July 9</b> <br>
<br> <b> July 9</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Extraction of BBa_J176171, BBa_K817002 P<i>fadBA</i> and pEGFP-N1 by miniprep<br>
+
·      Inoculation of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br>
-
·      Restriction of pEGFP-N1 Restriction digestion by Ase1 and BamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification<br>
+
·      Ethanol precipitation of pCEPcin<br>
-
·      Restriction of BBa_J176171 for P<i>fadBA</i> Ase1 and BamH1; FABP1 promoter by Xba1 and Not1<br>
+
·      Gel purification of digested BBa_E0240<br>
-
·      Ran gel for pEGFP-N1 and BBa_J176171 restriction products<br>
+
·      Gel check for BBa_E0240 insert and pSB3K3 backbone<br>
-
·      PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
+
·      PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br>
-
·      <a href="http://parts.igem.org/Part:BBa_J52034">BBa_J52034</a> (<i>fad</i>R) transformation<br>
+
·      Inoculation of pCEPcin<br>
 +
·      E0240-PcomX & PcomCDE: Digestion w/ EcoRI and PstI; Gel electrophoresis<br>
 +
·      J04450 (KAN): Gel purification<br>
 +
·      <i>comE<sup>D58E</sup></i>:Plasmid extraction; NanoDrop; Gel electrophoresis (not <i>comE</i>); Bacterial inoculation<br>
 +
 
<br> <b> July 10</b> <br>
<br> <b> July 10</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter<br>
+
·      Another PCR of <i>comW</i>-FLAG using Vent DNA Polymerase<br>
-
·      PCR for FABP1 promoter, PCR replication<br>
+
·      Gel check for <i>comW</i>-FLAG PCR product, PCR clean up<br>
-
·      BBa_J176171 vector dephosphorylation  by antartic phosphatase<br>
+
·      Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc<br>
-
·      Ligation of BBa_K817002 and P<i>fadBA</i> and BBa_J176171 <br>
+
·      Gel check and insert wanted present in several candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt), but no insert wanted present in any candidate colonies of ligated pSB1C3-K880005.comX-myc<br>
-
·      BBa_J52034 (<i>fad</i>R) inoculation<br>
+
·      Digestion of pCEPcin at EcoRI and SpeI, Xho and BamHI<br>
-
·     Digestion for BBa_K817002 P<i>fadBA</i> extraction<br>
+
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
-
Gel check for BBa_K817002 P<i>fadBA</i> extraction<br>
+
·      PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br>
 +
·   Ethanol precipitation of BBa_E0240 insert<br>
 +
·      Ligate E0240-PcomX & PcomCDE <br>
 +
·      Extract J04450 (KAN)<br>
 +
·      Restrict check & run mutagenesis <i>comE<sup>D58E</sup></i><br>
 +
 
<br> <b> July 11</b> <br>
<br> <b> July 11</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Gel purification for BBa_K817002 P<i>fadBA</i> extraction<br>
+
·      Digestion of <i>comW</i>-FLAG with XbaI and PstI, digestion clean up<br>
-
·      Gel check for FABP1 promoter PCR product<br>
+
·      Gel check for the PCR product obtained on July 10<br>
-
·      PCR clean-up<br>
+
·      Digestion of pCEPcin at NcoI and XhoI<br>
-
·      BBa_J52034 (<i>fad</i>R) miniprep<br>
+
·      Ethanol precipitation of pCEPcin<br>
-
·      BBa_J52034 restriction by Pst1 HF and Not1 HF<br>
+
·      Extract J04450 (KAN)<br>
-
·      PCR  for PPAR-alpha promoter<br>
+
·      Ligate pSB1C3 with <i>comE<sup>D58E</sup></i> <br>
-
Dry lab<br>
+
·      TransformPcomCDE<br>
-
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions<br>
+
·   <br>
-
 
+
-
<br> <b> July 12</b> <br>
+
-
Wet lab<br>
+
-
·  Digestion of FABP1 promoter PCR product<br>
+
-
·      Ran gel for PCR check of PPAR-alpha promoter<br>
+
-
·      Digest pEGFP-n1 for BBa_J52034 <i>fad</i>R<br>
+
-
·      Ran gel for pEGFP-n1 for BBa_J52034 <i>fad</i>R followed by gel purification<br>
+
-
·      BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and P<i>fadBA</i><br>
+
-
Dry Lab<br>
+
-
Primers design for pCMV cloning for <i>fad</i>R expression<br><br>
+
       </div>
       </div>
   </div>
   </div>
Line 438: Line 382:
       <a href="#" class="head">Week 3</a>
       <a href="#" class="head">Week 3</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>July 15</b> <br>
+
<br> <b> July 14</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation<br>
+
·      Ligation of <i>comW</i>-FLAG cut on XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut on SpeI and PstI<br>
-
·      Transformation of FABP1 promoter, EGFP, and BBa_J176171 ligation<br>
+
·      Transformation of ligation products<br>
-
·      FABP1 promoter+EGFP +BBa_J176171 ligation restriction check<br>
+
·      Inoculation of ligated dephosporylated pSB1C3-K880005.comX-myc<br>
 +
·      Digestion of pCEPcin at NcoI and XhoI<br>
 +
·      PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br>
 +
·      Gel purification of digested BBa_J04450<br>
 +
·      Inoculation of pCEPcin<br>
 +
·      PCR of <i>comE<sup>D58E</sup></i> with only 1 primer → check whether the primers are working or not<br>
 +
·      Ligation of pSB1C3-PcomX<br>
 +
·      Inoculation of pSB1C3-PcomCDE<br>
 +
·      Digest B0015<br>
 +
·      Digest <i>comE<sup>D58E</sup></i><br>
 +
·      Ligation of PCR product <i>comE</i> to pSB1C3<br>
-
<br><b>July 16</b> <br>
 
-
Wet Lab<br>
 
-
·      Miniprep for full construct of FABP1 promoter and pEGFP-N1<br>
 
-
·      <i>fad</i>R and pEGFP-N1 Backbone parts ligation <br>
 
-
·      Plasmids extraction for pCMV cloning for <i>fad</i>R<br>
 
-
·      BBa_K817002 P<i>fadBA</i> promoter extraction by EcoR1 and Pst1 HF<br>
 
-
·      BBa_J52034 restriction by EcoR1 and Pst1 HF<br>
 
-
·      Inoculations for full construct of FABP1 promoter and pEGFP-N1<br>
 
-
·      Streak colonies containing the right construct for FABP1 promoter<br>
 
-
<br><b>July 17</b> <br>
+
<br> <b> July 15</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep<br>
+
·      Digestion check of ligated pSB1C3-K880005.comX-myc<br>
-
·      Repeat <i>fad</i>R and P<i>fadBA</i> constructs<br>
+
·      Digestion of pSB1C3-K880005.comX-myc on SpeI and PstI, but no insert wanted present<br>
-
·      Digest pEGFP-n1 and BBa-J176171 for <i>fad</i>R<br>
+
·      PCR of comX using Vent DNA Polymerase, PCR clean-up<br>
-
·      Digestion for BBa_K817002 (P<i>fadBA</i>) extraction<br>
+
·      Colony PCR of pSB1C3-K880005.comX-myc using Taq DNA Polymerase<br>
-
·      Gel check and gel extraction for BBa_J176171 for <i>fad</i>R and BBa_K817002 (P<i>fadBA</i>) <br>
+
·      Gel check of pSB1C3-K880005.comX-myc PCR product (no PCR product present)<br>
 +
·      Inoculation of dephosporylated pSB1C3-K880005.comX-myc, dephosporylated pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Ethanol precipitation of pCEPcin<br>
 +
·      Gel check of the PCR product obtained on July 14<br>
 +
·   Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      PCR of 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> using Phusion polymerase<br>
 +
·      Plasmid extraction of pSB1C3-PcomCDE<br>
 +
·      Bacterial inoculation (5 for PcomCDE, 4 for PcomX)<br>
 +
·      Bacterial inoculation of B0015 (6 tubes)<br>
 +
·      Extract gel B0015 & <i>comE<sup>D58E</sup></i><br>
 +
·      K880005 dephosphorylation; Digestion<br>
 +
·   <br>
-
<br><b>July 18</b> <br>
+
<br> <b> July 16</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Gel purification for all digested products<br>
+
·      Digestion check of ligated pSB1C3-K880005.<i>comW</i>-FLAG<br>
-
·      FABP1 promoter digestion check for whole construct prior transfection<br>
+
·      Colony PCR of pSB1C3-K880005.<i>comW</i>-FLAG<br>
-
·      P<i>fadBA</i> vector de phosphorylation and ligation with EGFP and BBa_J176171<br>
+
·      Gel check of pSB1C3-K880005.<i>comW</i>-FLAG PCR product (no PCR product present)<br>
-
·      Transformation of P<i>fadBA</i>+EGFP+BBa_J176171<br>
+
·      PCR of <i>comW</i> using Vent DNA Polymerase (no PCR product present)<br>
 +
·      Digestion of comX on XbaI and PstI<br>
 +
·      Gel check of the PCR product obtained on July 15<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
 +
·      Digestion of pCEPcin2 at XhoI and SalI<br>
 +
·   PCR of 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> using Phusion polymerase<br>
 +
·   Inoculation of pCEPcin2 and pSB3K3-BBa_I20260<br>
 +
·      Plasmid extraction of Promoters and B0015<br>
 +
·      Colony PCR<br>
 +
·      Extract gel B0015 & <i>comE<sup>D58E</sup></i><br>
 +
·      Mutagenesis attempts for <i><i>comE</i></i><br>
-
<br><b>July 19</b> <br>
+
<br> <b> July 17</b> <br>
Wet lab<br>
Wet lab<br>
-
·      FABP1 promoter preparation for transfection<br>
+
·      PCR of comX-myc and comX using Vent DNA Polymerase<br>
-
·      Ligation of P<i>fadBA</i> and BBa_J176171 followed by transformation<br>
+
·      Gel check of comX-myc and comX PCR product, PCR clean-up<br>
-
·      PCR for PPAR-alpha promoter cloning<br>
+
·      PCR of <i>comW</i> using Vent DNA Polymerase<br>
-
Dry lab<br>
+
·      Gel extraction and gel purification of <i>comW</i> PCR product<br>
-
·      Design for characterization of the transfected cells with FABP1 construct<br>
+
·      Dephosporylation of BBa_K880005<br>
-
·      Review for Multiple Sites Mutagenesis<br>
+
·      Gel check of the PCR product obtained on July 16, all types of P<sub>combox</sub> shown positive result<br>
-
·      Ordered primers for pCMV cloning from pEGFP-N1<br><br>
+
·      PCR clean-up of P<sub>combox</sub><br>
 +
·      PCR product ligation with pSB1C3<br>
 +
·      Bacterial inoculation of B0015, <i>comE<sup>D58E</sup></i><br>
 +
·      Extract gel B0015 & <i>comE<sup>D58E</sup></i> - Digestion<br>
 +
·      Overnight Ligation<br>
 +
·   <br>
 +
<br> <b> July 18</b> <br>
 +
Wet lab<br>
 +
·      PCR of comX using Vent DNA Polymerase<br>
 +
·      Gel check of comX PCR product, PCR clean-up<br>
 +
·      Digestion of comX PCR product on XbaI and PstI, digestion clean-up<br>
 +
·      Digestion of comX-myc and <i>comW</i> PCR products, digestion clean-up<br>
 +
·      Gel extraction and gel purification of digested comX and comX-myc<br>
 +
·      Digestion of pCEPcin2 (at XhoI and SalI) and pSB3K3-BBa_I20260 (at XbaI and PstI)<br>
 +
·      Digestion of pCEPcin2 (at BamHI and NcoI)<br>
 +
·   PCR of 100bp P<sub>combox</sub> using Phusion polymerase<br>
 +
·      Gel check of the purified P<sub>combox</sub> obtained July 17<br>
 +
·   Gel check of the 100bp P<sub>combox</sub> (PCR product)<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
 +
·      Ligation of <i>comE<sup>D58E</sup></i><br>
 +
·      Gel extract <i>comE<sup>D58E</sup></i><br>
 +
·      BBa_B0015 Gel purification<br>
 +
·   <br>
 +
 
       </div>
       </div>
Line 485: Line 474:
       <a href="#" class="head">Week 4</a>
       <a href="#" class="head">Week 4</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>July 22</b> <br>
+
<br> <b> July 21</b> <br>
Wet lab<br>
Wet lab<br>
-
· FABP1 construct given for characterization and transfection<br>
+
·     Gel purification of comX and comX-myc<br>
-
· Further colony screening for FABP1 construct, inoculations<br>
+
·     Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br>
-
· Inoculation of P<i>fadBA</i> and BBa_J176171<br>
+
·     Ligation of <i>comW</i>-FLAG into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br>
-
· Ran gel for PPAR-alpha promoter PCR products<br>
+
·      Ligation of comX into dephosporylated pSB1C3-K880005 backbone with 1:3 and 1:5 ratio<br>
 +
·     Ligation of <i>comW</i> into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio<br>
 +
·      Colony PCR of pSB1C3-K880005.<i>comW</i>-FLAG using Taq DNA Polymerase<br>
 +
·      PCR of 100bp and 150bp P<sub>combox</sub> using Phusion polymerase<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
 +
·      Colony PCR of Promoters<br>
-
<br><b>July 23</b> <br>
 
-
Wet lab<br>
 
-
·  Primers arrival for pCMV cloning<br>
 
-
·  PCR for pCMV cloning<br>
 
-
·  Digestion and gel for construction check for FABP1 promoter<br>
 
-
·  P<i>fadBA</i> gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation<br>
 
-
·  Digestion of pEGFP-N1 for <i>fad</i>R, followed by gel check and extraction<br>
 
-
<br><b>July 24</b> <br>
+
<br> <b> July 22</b> <br>
Wet lab<br>
Wet lab<br>
-
·      DNA purification from gel extraction for digested pEGFP-N1<br>
+
·      Colony PCR of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG and use BBa_J04450 as the positive control using Taq DNA Polymerase (no PCR product present)<br>
-
·      Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter<br>
+
·      Inoculation of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG<br>
-
·      Ran gel for pCMV cloning from pEGFP-N1<br>
+
·      Gel check of the PCR product obtained on July 21<br>
-
·      PCR clean up for pCMV cloning from pEGFP-N1<br>
+
·      Oligo annealing of 67bp P<sub>combox</sub><br>
-
·      PCR for PPAR-alpha promoter with new primers using Taq polymerase<br>
+
·      Digestion of pSB3K3-BBa_I20260 at XbaI and PstI<br>
-
·      Transformation of mutagenesis products<br>
+
·      PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Phusion polymerase<br>
-
·      Inoculation of P<i>fadBA</i> ligation colonies<br>
+
·      Gel electrophoresis of colony PCR results<br>
 +
·      Mutagenesis of <i>comE<sup>D58E</sup></i><br>
 +
·   <br>
-
<br><b>July 25</b> <br>
+
<br> <b> July 23</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase<br>
+
·      Digestion check of comX, <i>comW</i>, comX-myc, <i>comW</i>-FLAG colony PCR products on XbaI and SpeI<br>
-
·      Minprep of P<i>fadBA</i>+EGFP+BBa_J176171 ligation colonies, followed by digestion check <br>
+
·      Inoculation of <i>comW</i>-FLAG<br>
 +
·      Gel check of PCR product obtained on July 22<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
 +
·      PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Phusion polymerase<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
 +
·      Gel electrophoresis of colony PCR results<br>
 +
·      Mutagenesis of <i>comE<sup>D58E</sup></i><br>
 +
·   <br>
-
<br><b>July 26</b> <br>
+
<br> <b> July 24</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter<br>
+
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and SpeI-HF<br>
-
·      Ran gel before parental string digestion of mutagenesis products<br>
+
·      Ligation of <i>comW</i>-FLAG into dephosporylated pSB1C3-K880005 backbone<br>
-
·      Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check<br>
+
·      Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone<br>
-
·      Plasmids preparation according to transfection requirements, construct and controls<br><br>
+
·      PCR of comX-myc and comX using Vent DNA Polymerase, gel extraction and gel purification<br>
 +
·      Digestion of comX-myc and comX on XbaI and PstI-HF<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Gel Check of PCR product obtained on July 23<br>
 +
·      Received pCEPcin2<br>
 +
·      Gel electrophoresis of colony PCR and mutagenesis results<br>
 +
·   <br>
 +
<br> <b> July 25</b> <br>
 +
Wet lab<br>
 +
·      Colony PCR of comX-myc using Taq DNA Polymerase<br>
 +
·      Gel check of comX-myc PCR product (no PCR product present)<br>
 +
·      Gel purification of digested pSB3K3 backbone<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Gel check on K880005<br>
 +
·      Transformation of ligated product (promoters+pSB1C3)<br>
 +
·   <br>
       </div>
       </div>
   </div>  
   </div>  
Line 528: Line 540:
       <a href="#" class="head">Week 5</a>
       <a href="#" class="head">Week 5</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>July 30</b> <br>
+
<br> <b> July 28</b> <br>
Wet lab<br>
Wet lab<br>
-
·      PPAR-alpha promoter PCR cloning with new primers using Vent polymerase<br>
+
·      Colony PCR of comX-myc using Taq DNA Polymerase<br>
-
·      Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1<br>
+
·      Colony PCR of <i>comW</i>-FLAG using Taq DNA Polymerase<br>
-
·      BBa_J176171 vector de phosphorylation, followed by ligation with EGFP using T4 Ligase, then transformation into E. Coli strain DH10b<br>
+
·      Inoculation of comX-myc and <i>comW</i>-FLAG<br>
-
·      PCR for PPAR-alpha promoter with new primers using Vent polymerase<br>
+
·      Digestion of pCEPcin2 at EcoRI and PstI, EcoRI and SpeI, NcoI and XhoI<br>
-
Dry lab<br>
+
·      Colony PCR of promoters-pSB1C3<br>
-
·      Discussion and re assessment of constructs related to pEGFP-N1<br>
+
·      Bacterial inoculation of K880005, <i>comE<sup>D58E</sup></i> <br>
 +
·      <br>
 +
·   <br>
 +
<br> <b> July 29</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br>
 +
·      Transformation of comX-myc and <i>comW</i>-FLAG<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Digestion of pCEPcin2 at EcoRI and PstI<br>
 +
·      Inoculation of pSB3K3-BBa_J04450<br>
 +
·      PCR of 100bp,150bp and 160bp P<sub>combox</sub> using Vent polymerase<br>
 +
·      Restrict check K880005 <br>
 +
·      Promoters colony PCR <br>
 +
·      PCR for <i>comE</i> fragments (mutagenesis) for Gibson Assembly<br>
-
<br><b>July 31</b> <br>
+
 
-
·      Ran gel check for PPAR-alpha promoter PCR cloning with new primers using Vent polymerase<br>
+
 
-
·      PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up<br>
+
<br> <b> July 30</b> <br>
-
·      PCR for PPAR-alpha promoter with reference primers using Vent polymerase<br>
+
Wet lab<br>
-
·      Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase<br><br>
+
·      Digestion check of comX-myc and <i>comW</i>-FLAG<br>
 +
·      Inoculation of comX-myc and <i>comW</i>-FLAG <br>
 +
·      Streaking plates of comX-myc and <i>comW</i>-FLAG<br>
 +
·      PCR clean-up of 100bp,150bp and 160bp P<sub>combox</sub><br>
 +
·      Digestion of pCEPcin2 at EcoRI and PstI<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      Restrict check K880005  <br>
 +
 
 +
<br> <b> July 31</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br>
 +
·      Redigest <i>comW</i>-FLAG on XmnI and NcoI<br>
 +
·      PCR sequencing for <i>comW</i> and comX<br>
 +
·      Ethanol precipitation for <i>comW</i> and comX<br>
 +
·      Digestion of pSB3K3-BBa_J04450 at XbaI and PstI<br>
 +
·      K880005 dephosphorylation & ligation<br>
 +
·      Promoters Colony PCR + Sequencing reaction<br>
 +
·      PCR for <i>comE<sup>D58E</sup></i> fragments (mutagenesis) for Gibson Assembly<br>
       </div>
       </div>
Line 560: Line 602:
-
<div id="satu" align="center"> <h2>August 2013</h2>
+
<div id="satu" align="center"> <h2>August 2014</h2>
-
    
+
   <div>
 +
      <a href="#" class="head">Week 1</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 1</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-BBa_J04450<br>
 +
·      Restrict check Promoters & Sequencing reaction<br>
 +
·      Transform K880005<br>
 +
·   <br>
 +
 
 +
      </div>
 +
    </div>
<div>
<div>
 +
      <a href="#" class="head">Week 2</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 4</b> <br>
 +
Wet lab<br>
 +
·      Digestion of comX-myc on EcoRI-HF and SpeI, gel extraction and gel purification<br>
 +
·      Ligation of BBa_B0015 cut on XbaI and PstI into pSB1C3-comX-myc backbone cut on SpeI and PstI<br>
 +
·      Transformation of pSB1C3-comX-myc.B0015 ligation product<br>
 +
·      Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br>
 +
·      Transformation of pSB1C3-B0015.comX-myc ligation product<br>
 +
·      Sequencing of comX-myc and <i>comW</i>-FLAG<br>
 +
·      Inoculation of comX-myc<br>
 +
·      Digestion of 100bp, 150bp, 160bp and 180bp P<sub>combox</sub> at XbaI and PstI<br>
 +
·      Ligation of 100bp and 160bp P<sub>combox</sub> with pSB1C3<br>
 +
·   Digestion of pCEPcin2 at EcoRI and PstI<br>
 +
·      PcomCDE Precipitation<br>
 +
·      PcomX Sequencing reaction & precipitation<br>
 +
·      Transform <i>comE<sup>D58E</sup></i>  <br>
 +
·      Gibson Assembly for <i>comE</i> and <i>comE<sup>D58E</sup></i> fragments<br>
 +
 +
<br><b>Aug 5</b> <br>
 +
Wet lab<br>
 +
·      Sequencing result failed<br>
 +
·      Transformation of pSB1C3-B0015.comX-myc ligation product failed<br>
 +
·      Digestion check of comX-myc on StyI<br>
 +
·      Colony PCR of comX-myc<br>
 +
·      Inoculation of comX-myc<br>
 +
·      Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
 +
·      Ligation of 150bp, 180bp and 249bp P<sub>combox</sub> with pSB1C3<br>
 +
·      Transformation of pSB1C3-150bp P<sub>combox</sub> , pSB1C3-180bp P<sub>combox</sub>  and pSB1C3-249bp P<sub>combox</sub><br>
 +
·      Gel purification of digested pSB1C3 backbone<br>
 +
·   Inoculation of pCEPcin2<br>
 +
·      Send to sequencing: PcomCDE and PcomX<br>
 +
·      Bacterial inoculation of <i>comE<sup>D58E</sup></i>  <br>
 +
·      Colony PCR to check pSB1C3-<i><i>comE</i></i> and pSB1C3-<i><i>comE</i><sup>D58E</sup></i><br>
 +
 +
 +
<br><b>Aug 6</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc on XbaI and PstI<br>
 +
·      Gel check of comX-myc PCR product (no PCR product present)<br>
 +
·      Sequencing of <i>comW</i>-FLAG using VF2 and VR<br>
 +
·      Digestion of comX-myc on StyI<br>
 +
·      Digestion of pCEPcin2 at EcoRI and EcoRV, EcoRI and PstI, EcoRI and SpeI, XhoI and NcoI<br>
 +
·      Gel purification of digested pSB1C3 backbone<br>
 +
·      Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
 +
·      Phosphorylation of 67bp P<sub>combox</sub> oligos<br>
 +
·      Oligo annealing for 67bp P<sub>combox</sub><br>
 +
·      Align sequence with expected: P<sub>comCDE</sub> and P<sub>comX</sub><br>
 +
 +
 +
<br><b>Aug 7</b> <br>
 +
Wet lab<br>
 +
·      Digestion of comX-myc on StyI<br>
 +
·      Digestion check of comX on SphI and NcoI<br>
 +
·      Digestion check of <i>comW</i> on XmnI and NcoI<br>
 +
·      Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
 +
·      Colony PCR of 100bp, 150bp, 160bp, 180bp and 249bp P<sub>combox</sub><br>
 +
·      Gel purification of digested pSB1C3 backbone<br>
 +
·      Precipitate after sequencing reaction PcomCDE<br>
 +
·      Make glycerol stock for PcomX<br>
 +
·      Run colony PCR for <i>comE<sup>D58E</sup></i><br>
 +
·   <br>
 +
<br><b>Aug 8</b> <br>
 +
Wet lab<br>
 +
·      Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI, gel extraction and gel purification<br>
 +
·      Digestion of <i>comW</i>-FLAG on SpeI and PstI-HF, digestion clean-up<br>
 +
·      Sequencing of comX-myc succeeded but mutation at EcoRI site<br>
 +
·      Digestion check of comX-myc at EcoRI-HF and PstI-HF<br>
 +
·      Ligation of comX and <i>comW</i> into dephosporylated pSB1C3-K880005<br>
 +
·      Transformation of comX and <i>comW</i> ligation product<br>
 +
·      Digestion of comX-myc at EcoRI-HF and SpeI-HF<br>
 +
·      Digestion of pCEPcin2 at EcoRI and EcoRV, XhoI and NcoI<br>
 +
·      Ligation of 67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp P<sub>combox</sub> with pSB1C3 backbone<br>
 +
·      Ligate with E0240 PcomX  <br>
 +
·      Colony PCR for <i>comE</i> mutant<br>
 +
·      Check sequence PcomCDE<br>
 +
·   <br>
 +
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 3</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 11</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of comX-myc and <i>comW</i>-FLAG on XbaI and PstI-HF<br>
 +
·      Gel purification of comX-myc digested on EcoRI-HF and SpeI<br>
 +
·      Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 180bp and 249bp) at XbaI and PstI<br>
 +
·      Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp)<br>
 +
·      Gel purification of digested pSB1C3 backbone<br>
 +
·   Digestion of pSB1C3-BBa_J04450 at XbaI and PstI<br>
 +
·      Extract pSB1C3 from J04450<br>
 +
·      Redo ligation part 1<br>
 +
·      Gibson assembly of <i><i>comE</i><sup>D58E</sup></i><br>
 +
·      Ligation of PCR product <i>comD</i> to pSB1C3<br>
 +
 +
<br><b>Aug 12</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.comX and pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF<br>
 +
·      Dilution of <i>comW</i>-FLAG<br>
 +
·      Re-digestion of BBa_B0015 on EcoRI-HF and XbaI, digestion clean-up<br>
 +
·      Re-ligation of pSB1C3-B0015.comX-myc<br>
 +
·      Transformation of pSB1C3-B0015.comX-myc ligation product<br>
 +
·      Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX-myc<br>
 +
·      Digestion of pCEPcin2 at EcoRI and EcoRV<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br>
 +
·      Ligate K880005 and <i>comE</i>(D58E)<br>
 +
·      Inoculate for glycerol stock (PcomX)<br>
 +
·   <br>
 +
<br><b>Aug 13</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-B0015.comX-myc and pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Inoculation of  pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI<br>
 +
·      Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      Check new dNTP<br>
 +
·      Restrict check of ligation product part 1<br>
 +
·      Colony PCR for checking part 2 ligation product and gibson assembly<br>
 +
·      Inoculation of PcomX for glycerol stock<br>
 +
·      <br>
 +
·   <br>
 +
<br><b>Aug 14</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Transformation of ligated product pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Dilution of <i>comW</i>-FLAG<br>
 +
·      Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Ligation of <i>comW</i>-FLAG into pSB1C3-B0015 backbone<br>
 +
·      Re-digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Gel check of the digested pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) obtained on July 13<br>
 +
·      Colony PCR of Colony PCR of pSB1C3-P<sub>combox</sub> (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      glycerol stock for PcomX<br>
 +
·      colony PCR ligation product part 1<br>
 +
·      colony PCR gibson assembly<br>
 +
·      Run gel for PCR products<br>
 +
·      Inoculation of <i>comE<sup>D58E</sup></i><br>
 +
·   <br>
 +
<br><b>Aug 15</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up<br>
 +
·      Colony PCR of <i>comW</i>-FLAG using Taq DNA Polymerase<br>
 +
·      Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF<br>
 +
·      Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI, digestion clean-up<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
 +
·      Overnight ligation of BBa_B0015 into pSB1C3-K880005.comX-myc backbone at 16°C<br>
 +
·      Transformation of pSB1C3-K880005.comX-myc.B0015 ligation product<br>
 +
·      Ligation of comX-myc into pSB1C3-B0015 backbone<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG and pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Preparation for submittion of samples to be sequenced<br>
 +
·      Inoculation of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp)<br>
 +
·      colony PCR for ligation product part 1<br>
 +
·      colony PCR for ligation product part 2<br>
 +
·      colony PCR for gibson assembly<br>
 +
·      Run gel for PCR products<br>
 +
·      <i>comE</i>-D58E plasmid extraction<br>
 +
·   <br>
 +
 +
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 4</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 18</b> <br>
 +
Wet lab<br>
 +
·      Transformation of ligated pSB1C3-K880005.comX-myc<br>
 +
·      Ligation of <i>comW</i>-FLAG into pSB1C3-K880005 backbone, comX-myc into pSB1C3-K880005 backbone, BBa_B0015 into pSB1C3-K880005.comX-myc backbone<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on SpeI-HF and PstI-HF<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
 +
·      Inoculation of pSB1C3-K880005.comX-myc and pSB1C3-B0015<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc on SpeI-HF and PstI-HF, digestion clean-up<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up<br>
 +
·      Sent out pSB1C3-P<sub>combox</sub> (100bp, 150bp and 180bp) samples for commercial sequencing<br>
 +
·   Digestion of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) at SpeI and PstI<br>
 +
·      Gel purification of digested pSB1C3-P<sub>combox</sub> (150bp, 160bp, 180bp and 249bp)<br>
 +
·   Inoculation of pSB1C3-BBa_E0240<br>
 +
·      Gel extraction and purification of E0240<br>
 +
·      Bacterial inoculation of PcomX (PcomCDE(3))<br>
 +
·      Gel extraction and purification of <i>comE</i> mutant<br>
 +
·   <br>
 +
 +
<br><b>Aug 19</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI, digestion clean-up<br>
 +
·      Digestion check of comX-myc on XbaI and PstI-HF<br>
 +
·      Ligation of pSB1C3-K880005.comX-myc cut on EcoRI-HF and SpeI-HF iinto pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG and pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Gel purification of digested pSB1C3-P<sub>combox</sub> (100bp)<br>
 +
·      Ligation of pSB1C3-P<sub>combox</sub> (100bp, 150bp, 160bp, 180bp and 249bp) with BBa_E0240 insert<br>
 +
·      Transformation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br>
 +
·      Digestion of pSB1C3-BBa_E0240 at XbaI and PstI<br>
 +
·      Gel purification of E0240 insert<br>
 +
·      Digest PcomX(PcomCDE(3))-pSB1C3<br>
 +
·      Make aliquotes for oligo - PcomCDE<br>
 +
·      PcomCDE Oligo annealing<br>
 +
·      Ligation of <i>comE</i> mutant insert with K880005<br>
 +
·      Ligation of <i>comE</i> insert with K880005<br>
 +
·      Gel electrophoresis of Gibson assembly mastermix checking<br>
 +
·      Plasmid extraction of J04450(CHL), PcomX(PcomCDE(3)) <br>
 +
·      Bacterial inoculation of <i>comE</i> WT, J04450 (CHL)<br>
 +
·   <br>
 +
<br><b>Aug 20</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on SphI and SpeI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc on XbaI and PstI<br>
 +
·      Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Gel purification of digested pSB1C3-BBa_E0240<br>
 +
·      Digestion of pSB1C3-180bp P<sub>combox</sub> at SpeI and PstI<br>
 +
·      Digestion clean-up of pSB1C3-180bp P<sub>combox</sub><br>
 +
·      Ligation of pSB1C3-180bp P<sub>combox</sub> with BBa_E0240 insert<br>
 +
·      Transformation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br>
 +
·      Colony PCR for ligated pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br>
 +
·   Inoculation of  pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp)<br>
 +
·      Ligation of E0240 insert with PcomX<br>
 +
·      Transformation [E0240-PcomCDE exp+ -ve, pSB1C3-PcomCDE exp+ -ve]<br>
 +
·      Ligation of <i>comE<sup>D58E</sup></i> insert with K880005<br>
 +
·      Gel extract <i>comE</i> WT<br>
 +
·      PCR check for <i>comE<sup>D58E</sup></i>-myc<br>
 +
·      Digest J04450(CHL) Gel extraction<br>
 +
·      Plasmid extraction of <i>comE</i> WT, J04450 (CHL)<br>
 +
·   <br>
 +
<br><b>Aug 21</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI<br>
 +
·      Colony PCR of pSB1C3-B0015 and reverse comX-myc using Taq DNA Polymerase<br>
 +
·      Digestion of <i>comW</i>-FLAG on NotI-HF<br>
 +
·      Digestion of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Transformation pSB1C3-K880005.<i>comW</i>-FLAG ligation product and pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) at NcoI<br>
 +
·      Colony PCR for ligated pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br>
 +
·      Inoculation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp)<br>
 +
·   Received sequencing result of pSB1C3-P<sub>combox</sub> (100bp, 150bp and 180bp)<br>
 +
·      Ligate PcomX with E0240 insert<br>
 +
·      PCR, Restrict check Part I (PcomCDE)<br>
 +
·      O/N Ligation w/ PcomCDE<br>
 +
·      Extract <i>comE</i> WT-pSB1C3<br>
 +
·      PCR and restrict check <i>comE<sup>D58E</sup></i>-myc<br>
 +
·      Glycerol stock for PcomX <br>
 +
·   <br>
 +
<br><b>Aug 22</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of <i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc.B0015 on EcoRI-HF and SpeI-HF<br>
 +
·      Inoculation pSB1C3-K880005.comX-myc.B0015<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI<br>
 +
·      PCR of pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) using Vent polymerase <br>
 +
·      Sent out pSB1C3-P<sub>combox</sub>-BBa_E0240 (160bp and 249bp) for commercial sequencing<br>
 +
·      Transformation of newly ligated (E0240)<br>
 +
·      Bacterial inoculation of checked colony (E0240)<br>
 +
·      O/N ligation w/ pSB1C3<br>
 +
·      Ligation <i>comE</i> WT-pSB1C3<br>
 +
·      Ligate <i>comE<sup>D58E</sup></i>-myc w/ K880005<br>
 +
·   <br>
 +
 +
 +
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 5</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 25</b> <br>
 +
Wet lab<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI
 +
Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
 +
Transformation of pSB1C3-K880005.<i>comW</i>-FLAG
 +
Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, gel extraction and gel purification
 +
Inoculation of pSB1C3-K880005.comX-myc.B0015 for glycerol stock<br>
 +
·      Received sequencing result of pSB1C3-P<sub>combox</sub> (160bp and 249bp)<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI<br>
 +
·      Digestion of pSB1C3-P<sub>combox</sub> (100bp, 150bp and 160bp, 180bp and 249bp) at XbaI and PstI<br>
 +
·      Gel purification of digested P<sub>combox</sub>-BBa_E0240 (100bp, 150bp and 180bp) insert<br>
 +
·      Ligation of PcomX-E0240<br>
 +
·      Digestion of PcomCDE-E0240<br>
 +
·      Colony PCR pSB1C3-PcomCDE, K880005-<i>comE</i> WT, K880005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
·   <br>
 +
<br><b>Aug 26</b> <br>
 +
Wet lab<br>
 +
·      Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, digestion clean-up<br>
 +
·      Ligation of Combox + GFP cut on XbaI and PstI-HF into pSB1C3-K880005.comX-myc.B0015 backbone cut on SpeI-HF and PstI-HF<br>
 +
·      Digestion of BBa_E0240 100 bp on XbaI and PstI-HF, gel extraction and gel purification<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
 +
·      Transformation of pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Ligation of comX, 100bp P<sub>combox</sub>-BBa_E0240 and pSB3K3 backbone<br>
 +
·      Gel pSB1C3-PcomCDE<br>
 +
·      PcomX-E0240 Colony PCR check<br>
 +
·      PcomCDE glycerol stock <br>
 +
·      Restrict check K880005-<i>comE</i> WT<br>
 +
·      Ligation w/ <i>comE</i> WT<br>
 +
·      Restrict check K880005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
 +
·   <br>
 +
<br><b>Aug 27</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF, gel extraction and gel purification<br>
 +
·      Sequencing of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG into pSB1C3-B0015 backbone and Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone <br>
 +
·      Transformation of pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Colony PCR of pSB1C3-P<sub>combox</sub>-BBa_E0240 (180bp and 249bp)<br>
 +
·      Ligation of comX, 100bp P<sub>combox</sub>-BBa_E0240 and pSB3K3 backbone<br>
 +
·      Inoculation of pSB1C3-P<sub>combox</sub>-BBa_E0240 (100bp, 180bp and 249bp), pSB1C3-P<sub>combox</sub> (160bp)<br>
 +
·      pSB1C3-PcomCDE<br>
 +
·      PcomCDE sequencing <br>
 +
·      PCR check K880005-<i>comE</i> WT<br>
 +
·      PCR check K880005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
·      Ligate K880005-<i>comE</i>-myc<br>
 +
 +
·   <br>
 +
<br><b>Aug 28</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF, gel extraction and gel purification<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
 +
·      Ligation of Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone<br>
 +
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 and pSB1C3-K880005.<i>comW</i>-FLAG<br>
 +
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG into pSB1C3-B0015 backbone<br>
 +
·      Restriction check of pSB1C3-P<sub>combox</sub>-BBa_E0240 (180bp and 249bp)<br>
 +
·      Colony PCR of pSB3K3-comX-100bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      pSB1C3-PcomCDE overnight ligation<br>
 +
·      PcomX-E0240 ligation<br>
 +
·      Gel extract I20260 and E0240<br>
 +
·      Glycerol stock for K880005-<i>comE</i> WT<br>
 +
·      Colony PCR for K880005-<i>comE<sup>D58E</sup></i>-myc  <br>
 +
·      Ligate K880005-<i>comE</i>-myc <br>
 +
·      PCR <i>comE<sup>D58E</sup></i>-myc<br>
 +
·      Ligation of Part 1 and Part 2<br>
 +
 +
·   <br>
 +
<br><b>Aug 29</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI-HF<br>
 +
·      Sent out pSB1C3-160bp P<sub>combox</sub> for commercial sequencing<br>
 +
·      Digestion of pSB1C3-160bp P<sub>combox</sub> at SpeI and PstI<br>
 +
·      Colony PCR for pSB3K3-comX-100bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      Ligation of pSB1C3-160bp P<sub>combox</sub> with BBa_E0240 insert<br>
 +
·      Transform pSB1C3-PcomCDE <br>
 +
·      Transform PcomX-E0240 <br>
 +
·      Ligate I20260 and E0240 <br>
 +
·      Ligate K880005-<i>comE</i> WT  <br>
 +
·      Ligate K880005-<i>comE</i>-myc<br>
 +
·      Ligate K880005-<i>comE<sup>D58E</sup></i>-myc  <br>
 +
·      Gel check <i>comE<sup>D58E</sup></i>-myc <br>
 +
·   <br>
 +
 +
 +
      </div>
 +
    </div>
 +
 +
 +
      </div>
 +
    </div>
 +
<!---- SSSSSSSS  EEEEEEEE  PPPPPPPP  TTTTTTTTTTTT  EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  RRRRRRRR    2 0 1 3-------->
 +
<!---- S          E          P      P      T        E          MM      MM  B      B  E          R    R 2 0 1 3-------->
 +
<!---- S          E          P      P        T        E          M M    M M  B      B  E          R    R 2 0 1 3-------->
 +
<!---- S          E          P      P        T        E          M  M  M  M  B      B  E          R  R 2 0 1 3-------->
 +
<!----     S          E          P      P        T        E          M  M M  M  B      B  E          R    R 2 0 1 3-------->
 +
<!----      SSSSSSSS  EEEEEEEE  PPPPPPPP        T        EEEEEEEE  M    M    M  BBBBBBBB    EEEEEEEEE  RRRRRRRR 2 0 1 3-------->
 +
<!----            S  E          P              T        E          M        M  B      B  E          R  R      2 0 1 3-------->
 +
<!----   S  E          P              T        E          M        M  B      B  E          R    R  2 0 1 3-------->
 +
<!----   S  E          P              T        E          M        M  B      B  E          R    R 2 0 1 3-------->
 +
<!----   S  E          p              T        E          M        M  B      B  E          R      R 2 0 1 3-------->
 +
<!---- SSSSSSSS  EEEEEEEE  P              T        EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  R  R 2 0 1 3-------->
 +
 +
<div id="satu" align="center"> <h2>September 2014</h2>
 +
 
 +
<div>
       <a href="#" class="head">Week 1</a>
       <a href="#" class="head">Week 1</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Aug xx</b> <br>
+
<br><b>Sept 1</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI-HF<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on XmnI<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Inoculation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Received the sequencing result of pSB1C3-160bp P<sub>combox</sub><br>
-
<br><b>Aug xx</b> <br>
+
·      Colony PCR: pSB1C3-PcomCDE, PcomX-E0240, I20260+Part2, E0240+Part2, E0240+Part2, K880005-<i>comE</i> WT, K990005-<i>comE</i>-myc, K990005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 2</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Ligation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP cut on EcoRI-HF and PstI-HF into pSB3K3 cut on EcoRI-HF and PstI-HF<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Re-digestion of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Restriction check of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240 at XmnI and NcoI<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Whole construct gel check <br>
-
<br><b>Aug xx</b> <br>
+
·      Restrict check PcomX-E0240<br>
 +
·      Restrict check Part 1 + Part 2<br>
 +
·      Restrict check I20260 and E0240 <br>
 +
·      Restrict check K880005-<i>comE</i> WT<br>
 +
·      Restrict check K880005-<i>comE</i>-myc<br>
 +
·      Restrict check K880005-<i>comE<sup>D58E</sup></i>-myc<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 3</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Streak plate for pSB1C3-K880005.comX-myc.B0015.Combox.GFP<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Restriction check of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Restrict check I20260-Part 2<br>
-
<br><b>Aug xx</b> <br>
+
·      Restrict check E0240-Part 2<br>
 +
·      Restrict check Whole construct<br>
 +
·      Restrict check -ve of whole construct ligation<br>
 +
·      Restrict check pSB1C3-PcomCDE<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 4</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI-HF, gel extraction and gel purification<br>
 +
·      Restriction of Combox + GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification<br>
 +
·      Ligation of Combox + GFP into pSB3K3 backbone cut on EcoRI-HF and PstI-HF<br>
 +
·      Transformation of pSB3K3-Combox.GFP ligation product<br>
 +
·      Digestion of pSB1C3-160bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      Ligation of 160bp P<sub>combox</sub>-BBa_E0240,comX and pSB3K3<br>
 +
·      Restrict check I20260-Part 2, E0240-Part 2, I20260-Part 2, whole construct, -ve of whole construct ligation<br>
 +
·      Restrict check PcomCDE-E0240<br>
 +
·      Restrict check I20260, E0240, Whole construct 3,4,5<br>
 +
·      Restrict check and colony PCR for pSB1C3-PcomCDE<br>
 +
·      Restrict check K880005+<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Restrict check  K880005+<i>comE</i>-C-myc<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 5</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Inoculation of pSB3K3-Combox.GFP<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Sequencing for pSB1C3-PcomCDE<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      <br>
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·   <br>
Line 596: Line 1,058:
       <a href="#" class="head">Week 2</a>
       <a href="#" class="head">Week 2</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Aug xx</b> <br>
+
<br><b>Sept 8</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion check of pSB3K3-Combox.GFP on XmnI<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Restrict check and plasmid PCR for PcomCDE-E0240<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Obtain sequencing results for PcomCDE-pSB1C3, whole construct (PcomX)<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      K880005-<i>comE</i>/<i>comE<sup>D58E</sup></i>-myc digest<br>
-
<br><b>Aug xx</b> <br>
+
·      Ligation of Part 1 and 2 (also I20260, E0240)<br>
 +
·      Transformation<br>
 +
 
 +
·   <br>
 +
 
 +
 
 +
<br><b>Sept 9</b> <br>
Wet lab<br>
Wet lab<br>
-
·     Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·   <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
 
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
<br><b>Sept 10</b> <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
-
<br><b>Aug xx</b> <br>
+
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Streak plate for pSB3K3-Combox.GFP<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Restrict check PcomCDE-E0240<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Obtain sequencing results for PcomCDE-pSB1C3<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Whole construct (PcomX)<br>
-
<br><b>Aug xx</b> <br>
+
·      K880005-<i>comE</i>/<i>comE<sup>D58E</sup></i>-myc send to sequencing<br>
 +
·      Ligation and transformation of Part 1 and 2 (also I20260, E0240)<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 11</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Restriction check of pSB3K3-comX-160bp P<sub>combox</sub>-BBa_E0240 at EcoRI and PstI<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Obtain sequencing results PcomCDE-pSB1C3<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Gel electrophoresis of colony PCR product from 10/9 PcomCDE-E0240<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Bacterial inoculation of checked products (for sequencing) and streak plates<br>
 +
·      Check colony fluorescene for whole construct (PcomX)<br>
 +
·      Gel extraction and purification of E0240 and I20260<br>
 +
·      Gel extraction and purification of PcomCDE-E0240<br>
 +
·      Streak plate for K880005-<i>comE</i>-C-myc<br>
 +
·   <br>
 +
 +
<br><b>Sept 12</b> <br>
 +
Wet lab<br>
 +
·      Restriction check of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 at EcoRI and PstI<br>
 +
·      Transformation of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240<br>
 +
·      Make glycerol stock for PcomCDE-pSB1C3<br>
 +
·      Gel electrophoresis of colony PCR product of PcomCDE-E0240 from 10/9; Bacterial inoculation of checked products (for sequencing); Streak plate<br>
 +
·      Check colony and fluorescence for whole construct (PcomX)<br>
 +
·      Streak plate for K880005-<i>comE</i>-C-myc<br>
 +
·   <br>
       </div>
       </div>
     </div>
     </div>
Line 626: Line 1,111:
       <a href="#" class="head">Week 3</a>
       <a href="#" class="head">Week 3</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Aug xx</b> <br>
+
<br><b>Sept 15</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      PCR check and inoculation of colonies of I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Restrict check, Gel extraction and Gel purification of pSB3K3 J04450 (CHL)<br>
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·   <br>
-
<br><b>Aug xx</b> <br>
+
 
 +
 
 +
<br><b>Sept 16</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion of pSB1C3-K880005.<i>comW</i>-FLAG on EcoRI-HF and SpeI<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·   <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
 
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
<br><b>Sept 17</b> <br>
-
<br><b>Aug xx</b> <br>
+
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion of pSB1C3-K880005.<i>comW</i>-FLAG on XbaI and PstI<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Ligation of pSB1C3-K880005.<i>comW</i>-FLAG cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Digestion check of  pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox on XmnI<br>
-
<br><b>Aug xx</b> <br>
+
·      Digestion check of Combox + GFP on XmnI and PstI<br>
 +
·      Restriction check of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 at NotI and PstI<br>
 +
·      PCR check and inoculation for I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Restrict check pSB3K3<br>
 +
·      Gel extraction and Gel purification of J04450 (CHL) insert X,S<br>
 +
·      Sequencing reaction for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 18</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Ligation of comX-myc into pSB1C3 cut on XbaI and PstI<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Colony PCR and inoculation of correct colonies for I20260/E0240-K880005-<i>comE</i>-C-myc, I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Gel extraction and gel purification for J04450 (CHL), J04450 (KAN)<br>
 +
·      Sequencing reaction and Restrict check for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 19</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 on XbaI and PstI<br>
 +
·      Colony PCR, inoculation for I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Miniprep for I20260/E0240-K880005-<i>comE</i>-C-myc<br>
 +
·      Gel extraction and gel purification J04450 (CHL), J04450 (KAN) XS<br>
 +
·      Sequencing reaction for K880005-<i>comE</i>-C-myc<br>
 +
·   <br>
Line 657: Line 1,162:
       <a href="#" class="head">Week 4</a>
       <a href="#" class="head">Week 4</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Aug xx</b> <br>
+
<br><b>Sept 22</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Inoculation of pSB1C3-K880005.<i>comW</i>-FLAG.B0015<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Ligation of pSB3K3 backbone with 160bp P<sub>combox</sub>-BBa_E0240<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Gel extraction and gel purification of I20260/E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc EP<br>
-
<br><b>Aug xx</b> <br>
+
·      Colony PCR and inoculation of I20260/E0240-K880005-<i>comE</i>-C-myc <br>
 +
·      Gel extraction and gel purification J04450 (CHL), J04450 (KAN)<br>
 +
·      Oligo annealing and overnight ligation w/ pSB1C3for PcomCDE<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 23</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion of pSB1C3-K880005.<i>comW</i>-FLAG.B0015 of EcoRI-HF and SpeI-HF<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Digestion of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX on EcoRI-HF and SpeI-HF, gel extraction and gel purification<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Ligation of pSB1C3-K880005.comX into pSB1C3-B0015 cut on EcoRI-HF and XbaI<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Transformation of pSB3K3-160bp P<sub>combox</sub>-BBa_E0240 ligated on Sept 22<br>
-
<br><b>Aug xx</b> <br>
+
·      Gel check and redo liagtion for I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br>
 +
·      Gel extraction and purification for I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc<br>
 +
·      Ethanol precipitation for sequencing, restrict check  K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·      Dephosphorylation and gel extraction and gel purification of J04450 (CHL) (X&S)<br>
 +
·      Gel extraction and Gel purification, dephosphorylation of J04450 (KAN) (E&P)<br>
 +
·      Oligo annealing, overnight ligation w/ pSB1C3 of PcomCDE<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 24</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion check of pSB1C3-K880005.comX.B0015 on XbaI and PstI, gel extraction and gel purification<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Inoculation of bacterial colonies carrying pSB3K3-160bp P<sub>combox</sub>-BBa_E0240<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Restrict check, gel extraction and purification, Ligation w/ pSB3K3 I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-·      myc, E0240-K880005-<i>comE</i>-C-myc <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Obtain sequencing results for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
-
<br><b>Aug xx</b> <br>
+
·      Transform pSB1C3-PcomCDE<br>
 +
·   <br>
 +
 
 +
<br><b>Sept 25</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Restrcition digestion of ligated pSB3K3-160P<sub>combox</sub>-BBa_E0240<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Restrict check, gel extraction and purification, Ligation w/ pSB3K3, I20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Obtain sequencing results for K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·      Colony PCR w/ ‘NEW FPcomCDE’ and VR, inoculation of  pSB1C3-PcomCDE<br>
 +
 
 +
·   <br>
 +
 
 +
<br><b>Sept 26</b> <br>
 +
Wet lab<br>
 +
·      Transformation of pSB3K3-160P<sub>combox</sub>-BBa_E0240 sample1<br>
 +
·      Streak plate for pSB3K3-BBa_I20260 and pSB3K3-BBa_E0240<br>
 +
·      Gel extraction and purificationI and ligation20260-K880005-<i>comE<sup>D58E</sup></i>-N-myc, E0240-K880005-<i>comE<sup>D58E</sup></i>-N-myc, I20260-K880005-<i>comE</i>-C-myc, E0240-K880005-<i>comE</i>-C-myc<br>
 +
·      Restrict check K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·      Inoculation for sequencing K880005-<i>comE<sup>D58E</sup></i>-N-myc, K880005-<i>comE</i>-C-myc<br>
 +
·      Transformation of pSB1C3-PcomCDE<br>
 +
 
 +
·   <br>
Line 688: Line 1,222:
       <a href="#" class="head">Week 5</a>
       <a href="#" class="head">Week 5</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Aug xx</b> <br>
+
<br><b>Sept 29</b> <br>
Wet lab<br>
Wet lab<br>
-
·     Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·   <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
 
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
 
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
<br><b>Sept 30</b> <br>
-
<br><b>Aug xx</b> <br>
+
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      Digestion of  RBS+comX-myc+B0015 on XbaI and PstI<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      Ligation of PcomCDE-E0240, both that day and overnight<br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      Transformation<br>
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·   <br>
-
<br><b>Aug xx</b> <br>
+
 
 +
 
 +
      </div>
 +
    </div>
 +
</div>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<h3>Riboregulator</h3>
 +
<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.7.1/jquery.min.js"></script>
 +
  <script>
 +
    $(document).ready(function(){
 +
      $('.head').each(function(){
 +
        var $content = $(this).closest('div').find('.content');
 +
        $(this).click(function(e){
 +
          e.preventDefault();
 +
          $content.not(':animated').slideToggle();
 +
    });
 +
});
 +
});
 +
  </script>
 +
  <style>
 +
    * { margin:0; padding:0; } /* a simple reset */
 +
 +
   
 +
 +
    .head {
 +
display:block;
 +
background:#FF9900;
 +
border-radius:2px;
 +
margin-bottom:5px;
 +
align:left;
 +
width:600px;
 +
height: 40px;
 +
z-index:-3;
 +
font-family:"Trebuchet MS", Helvetica, sans-serif;
 +
font-size: 35px;
 +
text-align: left;
 +
}
 +
 +
    .content {
 +
display:none;
 +
background:#8FF5D5;
 +
border-radius:10px;
 +
width:60%;
 +
margin-bottom:5px;
 +
align:center;
 +
z-index:-3;
 +
padding-left:100px;
 +
padding-right:100px;
 +
font-family:"Trebuchet MS", Helvetica, sans-serif
 +
}
 +
 +
   
 +
 
 +
  </style>
 +
 
 +
<div id="flight">
 +
<!--   J   U       U N       N EEEEEEEEEE   2 0 1 3---------------->
 +
<!--   J   U       U NN       N E   2 0 1 3---------------->
 +
<!--   J   U       U N N       N E   2 0 1 3---------------->
 +
<!--   J   U       U N  N   N E   2 0 1 3---------------->
 +
<!--   J   U       U N  N   N E   2 0 1 3---------------->
 +
<!--       J   U       U N    N   N EEEEEEEEEE    2 0 1 3---------------->
 +
<!--   J   U       U N    N   N E   2 0 1 3---------------->
 +
<!-- J   J   U       U N      N  N E       2 0 1 3---------------->
 +
<!-- J   J   U       U   N     N  N E   2 0 1 3---------------->
 +
<!-- J   J   U       U   N     N N E   2 0 1 3---------------->
 +
<!-- JJJJJJJJJJJJ   UUUUUUUUUUUU N       N EEEEEEEEEEE   2 0 1 3---------------->
 +
 
 +
 
 +
<!--   J   U       U L       Y            Y    2 0 1 3-------->
 +
<!--   J   U       U L           Y          Y 2 0 1 3-------->
 +
<!--   J   U       U L Y        Y 2 0 1 3-------->
 +
<!--   J   U       U L  Y      Y 2 0 1 3-------->
 +
<!--   J   U       U L                Y     Y        2 0 1 3-------->
 +
<!--       J   U       U L        Y  Y 2 0 1 3-------->
 +
<!--   J   U       U L            Y 2 0 1 3-------->
 +
<!-- J   J   U       U L    Y 2 0 1 3-------->
 +
<!-- J   J   U       U   L   Y 2 0 1 3-------->
 +
<!-- J   J   U       U   L   Y 2 0 1 3-------->
 +
<!-- JJJJJJJJJJJJ   UUUUUUUUUUUU LLLLLLLLLLLL     Y 2 0 1 3-------->
 +
 
 +
 
 +
 
 +
<div id="satu" align="center"> <h2><center>July 2014</center></h2>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 1</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br> <b> July 7 </b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. Oligos phosphorylation <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. ligation of TAs and CRs <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. Colony PCR <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. Digest Receptor Backbone pSB1C3 <br>
-
<br><b>Aug xx</b> <br>
+
. Agar-LB plates <br>
 +
. Inoculation for transformed Biobricks <br>
 +
. Primer Design for TA construct Gibson Assembly <br>
 +
. Gel check for the colony PCR <br>
 +
. P<sub>bad</sub> PCR amplification <br>
 +
 
 +
 
 +
<br> <b> July 8 </b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. Transformation of ligated TAs and CRs <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. Miniprep previous TAs CRs constructs <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. restriction check previous TAs CRs constructs <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. Colony PCR for CRs and TAs <br>
 +
. Re-ligation of TAs and CRs <br>
 +
. P<sub>bad</sub> PCR clean up and Digestion <br>
 +
. Ligation of P<sub>bad</sub> with pSB1C3 <br>
 +
. Sequencing preparation of P<sub>bad</sub> <br>
 +
 
 +
 
 +
<br> <b> July 9</b> <br>
 +
Wet lab<br>
 +
. Transformation of Re-ligated TAs and CRs <br>
 +
. Colony PCR <br>
 +
. Gibson assembly master mix preparation <br>
 +
 
 +
<br> <b> July 10</b> <br>
 +
Wet lab<br>
 +
. Sequencing preparation of P<sub>bad</sub> <br>
 +
. Miniprep previous TAs CRs constructs <br>
 +
. restriction check previous TAs CRs constructs <br>
 +
. Colony PCR <br>
 +
. Inoculation for glycerol stocks <br>
 +
. Digest Receptor Backbone , digestion of BBa_ BBa_E0240 with EcoR1-HF and Xbal and gel purification <br>
 +
 
 +
<br> <b> July 11</b> <br>
 +
Wet lab<br>
 +
. P<sub>bad</sub> digestion and gel purification, Digestion of P<sub>bad</sub>-pSB1C3 with EcoR1-HF and Spe1 and gel purification <br>
 +
. Ligation of P<sub>bad</sub> and BBa_E0240 <br>
 +
. Overlapping PCR design <br>
 +
. Gibson assembly master mix preparation <br>
 +
. Glycerol stock <br>
 +
. Competent cells preparations for DH5alpha and DH10B <br>
 +
. Run gel for yesterday's restriction check <br>
 +
. Miniprep BBa_E0240, mRFP, BBa_B0015 <br>
 +
. Colony PCR <br>
 +
. One full set re-ligation (short time) <br>
 +
. Miniprep BBa_I13401 and BBa_K592009 <br>
 +
 
 +
      </div>
 +
  </div>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 2</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br> <b> July 14 </b> <br>
 +
Wet lab<br>
 +
. Gibson assembly master mix preparation <br>
 +
. PCR <br>
 +
. Ligation of P<sub>bad</sub> an BBa_E0240 <br>
 +
. Competent cells preparations for DH5alpha and DH10B <br>
 +
. Prepare receptor backbone for oligo anealing, pSB1C3-BBa_J04450 (EcoRI, SpeI) <br>
 +
. One full set of re-ligation (short time) <br>
 +
. One full set of re-ligation (O/N) <br>
 +
. Preparations for P<sub>bad</sub> re-Characterization <br>
 +
 
 +
 
 +
<br> <b> July 15 </b> <br>
 +
Wet lab<br>
 +
. Inoculation for P<sub>bad</sub> -GFP-TT <br>
 +
. (2nd time) Colony PCR using VF2 and VR <br>
 +
. Design primer for P<sub>bad</sub> promoter sequencing <br>
 +
. Gibson assembly master mix preparation <br>
 +
. Preparations for P<sub>bad</sub>  re-characterization <br>
 +
. Competent cells preparations for DH5alpha and DH10B <br>
 +
. One full set re-ligation (O/N) <br>
 +
. Prepare receptor backbone for oligo anealing <br>
 +
 
 +
 
 +
<br> <b> July 16</b> <br>
 +
Wet lab<br>
 +
. Gibson assembly master mix preparation <br>
 +
. Preparations for P<sub>bad</sub> re-characterization <br>
 +
. Competent cells preparations for DH5alpha and DH10B <br>
 +
. Prepare receptor backbone for oligo anealing <br>
 +
. inoculate successful oligo annealing colonies <br>
 +
. Oligo annealing products C. PCR? <br>
 +
 
 +
<br> <b> July 17</b> <br>
 +
Wet lab<br>
 +
. Gibson assembly master mix preparation <br>
 +
. P<sub>bad</sub> re-characterization <br>
 +
. Miniprep CRs TAs <br>
 +
. Restriction check CRs,TAs <br>
 +
. Oligo annealing products C. PCR <br>
 +
. Prepare receptor backbone for oligo anealing <br>
 +
. Restrict CRs plasmids for GFP.dTT <br>
 +
. Restrict TAs plasmids for dTT <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Restrict dTT for TAs <br>
 +
. Ligate CR.GFP.dTT <br>
 +
. Ligate TA.dTT <br>
 +
. Transform Ligated CR.GFP.dTT <br>
 +
. Transform Ligated TA.dTT <br>
 +
. Transform-Ligate Annealed oligos <br>
 +
 
 +
<br> <b> July 18</b> <br>
 +
Wet lab<br>
 +
. Gibson assembly for amilCP <br>
 +
. P<sub>bad</sub>  re-characterization <br>
 +
. Colony PCR <br>
 +
. Prepare receptor backbone for oligo anealing <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Restrict dTT for TAs <br>
 +
. M9 mininaml medium preparation <br>
 +
. Colony PCR <br>
 +
. plate streaking for P<sub>bad</sub>-BBa_E0240 <br>
 +
. inoculation of TAs and CRs <br>
 +
 
 +
<br> <b> July 19</b> <br>
 +
Wet lab<br>
 +
. P<sub>bad</sub> re-characterization <br>
 +
 
 +
 
 +
      </div>
 +
  </div>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 3</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br> <b> July 21 </b> <br>
 +
Wet lab<br>
 +
. Gibson assembly for amilCP <br>
 +
. Miniprep CRs TAs <br>
 +
. restriction check pSB1C3-TAs/CRs constructs <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Restrict P<sub>bad</sub> for TAs <br>
 +
. Restrict BBa_B0015 EcoR1 and XbaI <br>
 +
. BBa_J04450 backbone extraction <br>
 +
. Colony PCR <br>
 +
. inoculation of TAs and CRs <br>
 +
. prepare P<sub>bad</sub> L(+) A. concentration gradients for characterization <br>
 +
. Restrict CRs <br>
 +
. Restrict TAs-Version1 <br>
 +
. Restrict TAs-Version2 <br>
 +
. Ligate CR.GFP.dTT <br>
 +
. Ligate TA.dTT <br>
 +
. Transform P<sub>bad</sub>.TA <br>
 +
. Transform CR.GFP.dTT <br>
 +
. Transform Gibson assembly <br>
 +
 
 +
 
 +
<br> <b> July 22 </b> <br>
 +
Wet lab<br>
 +
. Colony PCR <br>
 +
. Miniprep CRs TAs <br>
 +
. restriction check pSB1C3-TAs/CRs constructs <br>
 +
. Restrict BBa_B0015-Version2 <br>
 +
 
 +
 
 +
<br> <b> July 23</b> <br>
 +
Wet lab<br>
 +
. Inoculation candidate colony or construct <br>
 +
. Miniprep and restriction check inoculations <br>
 +
. Gibson assembly check <br>
 +
. Gibson PCR <br>
 +
. Teach inoue competent cells to main project <br>
 +
. Miniprep of BBa_B0015, P<sub>bad</sub>-BBa_E0240 <br>
 +
. glycerol stock (P<sub>bad</sub>-BBa_E0240) <br>
 +
. Re-ligation of oligo annealing O/N <br>
 +
 
 +
<br> <b> July 24</b> <br>
 +
Wet lab<br>
 +
. miniprep candidate colony or construct <br>
 +
. restriction check pSB1C3-TAs/CRs constructs <br>
 +
. Re-Re-ligation transformation <br>
 +
. Re-Re-ligation transformation V.X <br>
 +
. Gibson assembly check <br>
 +
 
 +
<br> <b> July 25</b> <br>
 +
Wet lab<br>
 +
. Certification day (safety evaluation) <br>
 +
 
 +
      </div>
 +
  </div>
 +
 
 +
  <div>
 +
      <a href="#" class="head">Week 4</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br> <b> July 28 </b> <br>
 +
Wet lab<br>
 +
. oligo phosphorylation <br>
 +
. oligo annealing <br>
 +
. ligation of annealed oligos O/N <br>
 +
. gel for C. PCR <br>
 +
. receptor backbone for annealed oligos <br>
 +
. inoculate last ligation <br>
 +
. Check past locks/keys status <br>
 +
. P<sub>bad</sub> sequencing <br>
 +
. transformation of some of the existing riboregulator Biobricks <br>
 +
. Benchling Check <br>
 +
 
 +
 
 +
<br> <b> July 29 </b> <br>
 +
Wet lab<br>
 +
. Miniprep and restriction check inoculations <br>
 +
. restriction check pSB1C3-TAs/CRs constructs <br>
 +
. Inoculation of CR and TA from distribution kit <br>
 +
. prepare BBa_I20260 in pSB1C3 <br>
 +
. prepare Side project Buffer <br>
 +
. prepare side project gel red 200:1 <br>
 +
. Check digested GFP <br>
 +
. Check digested BBa_B0015 <br>
 +
. ligate oligos into GFP <br>
 +
. ligate oligos into BBa_B0015 <br>
 +
. prepare M9 for characterization <br>
 +
. transform our P<sub>bad</sub> into DH5alpha <br>
 +
. prepare M9 medium <br>
 +
. transformation of Clon001-010 in pSB1C3 ( with a control) <br>
 +
 
 +
 
 +
<br> <b> July 30</b> <br>
 +
Wet lab<br>
 +
. transformation of CLON001-010 in pSB1C3 (BBa_B0015, BBa_I13401) <br>
 +
. C. PCR for yesterday transformations (CLON001-010) <br>
 +
. If construct after gel check inoculation, restriction, ligation <br>
 +
. prepare BBa_I20260 in pSB1C3 <br>
 +
. transform our P<sub>bad</sub>into DH5alpha <br>
 +
. prepare M9 for characterization <br>
 +
. Teach inoue competent cells to main project <br>
 +
 
 +
<br> <b> July 31</b> <br>
 +
Wet lab<br>
 +
. Try Gibson assembly mastermix <br>
 +
. Digest Biobrick TA and CR <br>
 +
. PCR clean up for existing CR and TA Biobricks <br>
 +
. run gel for yesterday's colony PCR <br>
 +
. check dNTPs <br>
 +
. Sequencing preparation for possible construct <br>
 +
. colony PCR for 5th <br>
 +
. inoculate candidates <br>
 +
. prepare BBa_I20260 in pSB1C3 <br>
 +
 
 +
 
 +
      </div>
 +
  </div>
 +
</div>
 +
<!------      AAAAAAAAAAAA    U        U  GGGGGGGGGGGG    U          U    SSSSSSSSSSSSS    TTTTTTTTTTTTTT      2 0 1 3---------------->
 +
<!------      A          A    U        U  G              U          U    S                      T      2 0 1 3---------------->
 +
<!------      A          A    U        U  G              U          U    S                  T      2 0 1 3---------------->
 +
<!------      A      A   U        U  G              U          U    S                  T        2 0 1 3---------------->
 +
<!------      A      A   U     U  G              U          U    S                  T        2 0 1 3---------------->
 +
<!------      AAAAAAAAAAAA   U        U  G              U          U    SSSSSSSSSSSSS          T        2 0 1 3---------------->
 +
<!------      A        A   U        U  G    GGGGGGG    U          U                S          T        2 0 1 3---------------->
 +
<!------      A        A   U        U G      G  G    U          U                S          T        2 0 1 3---------------->
 +
<!------      A        A   U        U G      G  G    U          U                S          T 2 0 1 3---------------->
 +
<!------      A        A   U        U G          G    U          U                S        T 2 0 1 3---------------->
 +
<!------      A        A   UUUUUUUUUUU  GGGGGGGGGGGG    UUUUUUUUUUUUU    SSSSSSSSSSSSS        T        2 0 1 3---------------->
 +
 
 +
 
 +
<div id="satu" align="center"> <h2>August 2014</h2>
 +
 
 +
<div>
 +
      <a href="#" class="head">Week 1</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 1</b> <br>
 +
Wet lab<br>
 +
. colony PCR for 5th <br>
 +
. Digest Biobrick TA and CR <br>
 +
. short ligation for TAs CRs <br>
 +
. Transform TAs, CRs <br>
 +
. inoue Buffer <br>
 +
. inoculations if possible <br>
 +
. miniprep CLON005 <br>
 +
. Digest 005 Spe1 and Pst1 <br>
 +
. ligate CLON 005 with BBa_B0015 <br>
 +
. prepare backbone : BBa_B0015 E, X /// BBa_I13401 E,X /// BBa_J23102 X, P <br>
 +
. Ligate P<sub>bad</sub> with Key1 dTT <br>
 +
. digest P<sub>tet</sub> lock 1 with S and P <br>
 +
 
 +
<br><b>Aug 2</b> <br>
 +
Wet lab<br>
 +
. Devise a general blue print for the catalog and feature page <br>
       </div>
       </div>
     </div>
     </div>
 +
<div>
 +
      <a href="#" class="head">Week 2</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 4</b> <br>
 +
Wet lab<br>
 +
. Mini prep 001-010 <br>
 +
. Restrict TAs <br>
 +
. restrict CRs <br>
 +
. Restrict P<sub>bad</sub> for TAs <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Restrict BBa_B0015 <br>
 +
. Ligate P<sub>bad</sub>.TA <br>
 +
. ligate CR.GFP.dTT <br>
 +
. Ligate P<sub>bad</sub>.dTT <br>
 +
. Transform TAs, CRs <br>
 +
. inoculate 001-010 <br>
 +
. Sequence 001-010 <br>
 +
. inoculate BBa_B0015 <br>
 +
. inoculate constitutive promoter <br>
 +
. inoculate P<sub>bad</sub> with key 1 dTT <br>
 +
. Competent cells preparations DH10B <br>
 +
. Prepare for functional assay of P<sub>bad</sub> promoter <br>
 +
. Prepare M9 minimal Medium <br>
 +
. make a second list of oligos to be sent out <br>
 +
. Test out GA mastermix efficiency <br>
 +
. Ligate TAs oligo with BBa_B0015 <br>
 +
. inoue Buffer <br>
 +
. Sequence 001-010 <br>
 +
 +
<br><b>Aug 5</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for TA.dTTs <br>
 +
. inoculate TA.dTT <br>
 +
. ligation of P<sub>bad</sub> and key3 <br>
 +
. Restrict TAs <br>
 +
. restrict CRs <br>
 +
. Restrict P<sub>bad</sub> for TAs <br>
 +
. Restrict GFP.dTT for CRs <br>
 +
. Ligate P<sub>bad</sub>.TA <br>
 +
. ligate CR.GFP.dTT <br>
 +
. Ligate P<sub>bad</sub>.dTT <br>
 +
. Transform TAs, CRs <br>
 +
. Prepare M9 medium <br>
 +
. Prepare inoculations for P<sub>bad</sub> characterization <br>
 +
. Sequecing preparation <br>
 +
. Digest P<sub>tet</sub> lock 1 with S and P <br>
 +
. Glycerol stock for 001-010 <br>
 +
. Inoue competent cells <br>
 +
. "Mini Prep pSB1C3-PCR P<sub>bad</sub><br>
 +
 +
<br><b>Aug 6</b> <br>
 +
Wet lab<br>
 +
. Miniprep TA.dTT <br>
 +
. Restriction check for TA.dTT <br>
 +
. Miniprep TAs CRs <br>
 +
. Restrict TAs <br>
 +
. Restrict CRs <br>
 +
. Restrict BBa_B0015 for TAs <br>
 +
. Colony PCR for TA.dTTs <br>
 +
. Oligo annealing for TA.dTT <br>
 +
. Inoue competent cells <br>
 +
. Sequencing results <br>
 +
. Ligate P<sub>bad</sub> with Key1 dTT <br>
 +
. Ligate P<sub>tet</sub> Lock 1 (GFP dTT) <br>
 +
. Send PCR P<sub>bad</sub> for sequencing <br>
 +
. Assay P<sub>bad</sub> promoter <br>
 +
 +
<br><b>Aug 7</b> <br>
 +
Wet lab<br>
 +
. Gel check for C. PCR <br>
 +
. inoculate possible TA.dTT <br>
 +
. Ligation of TA and dTT (4TAs) <br>
 +
. Ligation of P<sub>bad</sub> and key dTT <br>
 +
. Gel check for TAs CRs <br>
 +
. competent cells <br>
 +
. miniprep P<sub>bad</sub>-dTT <br>
 +
. miniprep P<sub>bad</sub>-key3 <br>
 +
. Colony PCR for P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3 <br>
 +
. Restriction of P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3 <br>
 +
. prepare backbone and or insert if any // and restriction check <br>
 +
. Preparation for P<sub>bad</sub> promoter characterization <br>
 +
. Colony PCR for P<sub>bad</sub> with Key 1 dTT <br>
 +
. Colony PCR for P<sub>tet</sub> Lock 1 GFP dTT <br>
 +
. inoculation of ligated DNA <br>
 +
 +
<br><b>Aug 8</b> <br>
 +
Wet lab<br>
 +
. digestion of P<sub>bad</sub>-key3 <br>
 +
. Restriction check for P<sub>bad</sub>-dTT and P<sub>bad</sub>-key3-DTT <br>
 +
. ligation of P<sub>bad</sub>-key3-dTT <br>
 +
. Miniprep for CLON007(J), CLON 010(I) and CLON (G) <br>
 +
. Miniprep P<sub>bad</sub> Key 1 dTT <br>
 +
. Miniprep P<sub>tet</sub> Lock 1 GFP dTT <br>
 +
. Restriction Check for the key 1 and lock 1 mentioned above <br>
 +
. Prepare digestion of key 1 and lock 1 for ligation <br>
 +
. Transform BBa_J13002 <br>
 +
. Restriction check CLON007(J), CLON 010(I) and CLON (G) <br>
 +
. digestion of CLON007(J), CLON 010(I) and CLON (G) <br>
 +
. ligation of CLON007(J), CLON 010(I) and CLON (G) with P<sub>bad</sub> <br>
 +
. C. PCR for CLON 022-025 TAs.dTT <br>
 +
. ligation of P<sub>bad</sub> and TAs <br>
 +
. ligation of GFP and CRs <br>
 +
. Inoue competent cells <br>
 +
. P<sub>bad</sub> promoter characterization <br>
 +
 +
<br><b>Aug 9</b> <br>
 +
Wet lab<br>
 +
. transformation , all in CHL, overnight ligation 16C <br>
 +
. Colony PCR gel check <br>
 +
. Check competent cells <br>
 +
 +
<br><b>Aug 10</b> <br>
 +
Wet lab<br>
 +
. Restriction check for TAs.dTT <br>
 +
. Gel check for C. PCR TAs.dTT <br>
 +
 +
       </div>
       </div>
     </div>
     </div>
-
</div>
+
<div>
 +
      <a href="#" class="head">Week 3</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 11</b> <br>
 +
Wet lab<br>
 +
. Restriction check for TAs.dTT <br>
 +
. Gel check for C. PCR TAs.dTT <br>
 +
. Colony PCR for CR.GFP.dTTs <br>
 +
. Colony PCR for P<sub>bad</sub>.TAs <br>
 +
. Inoculate CR.GFP.dTTs <br>
 +
. Inoculate P<sub>bad</sub>.TAs <br>
 +
. Restrict TA.dTTs <br>
 +
. Ligate P<sub>bad</sub>.TA.dTTs <br>
 +
. Transform P<sub>bad</sub>.TA.dTTs <br>
 +
. Restriction check for P<sub>tet</sub> Lock 1 GFP dTT and P<sub>bad</sub> Key 1 dTT <br>
 +
. Transform 2014 Kit plate 4 well GF <br>
 +
. Transformation of restricted checked DNA <br>
 +
. inoculate BBa_J13002 <br>
 +
. inoculate more P<sub>bad</sub> dTTs <br>
 +
. sequencing PCR for MCR, MTA <br>
 +
. transformation for backbone with KAN resistant <br>
 +
. inoculation B0034 from glycerol stock B0034 <br>
 +
. restriction check <br>
 +
. Ligation of P<sub>bad</sub>-key3 into BBa_B0015-pSB1AK3 plasmid <br>
 +
. inoculation of P<sub>bad</sub>-key3 <br>
 +
 
 +
<br><b>Aug 12</b> <br>
 +
Wet lab<br>
 +
. transformation P<sub>bad</sub> -TA <br>
 +
. Gel for TA.dTTs <br>
 +
. GEl for C. PCR for CR.GFP.dTT <br>
 +
. Digestion of PCR P<sub>bad</sub> <br>
 +
 
 +
<br><b>Aug 13</b> <br>
 +
Wet lab<br>
 +
. miniprep CR.GFP.dTTs <br>
 +
. miniprep P<sub>bad</sub> <br>
 +
. restriction check CR.GFP.dTTs <br>
 +
. inoculate TA.dTTs <br>
 +
. Gel extraction-purification of constitutive promoter <br>
 +
. Ligate CR.GFP.dTTs into constitutive promoter <br>
 +
. transform, P<sub>bad</sub>.Key3 <br>
 +
. transform constitutive promoter.CR.GFP.dTTs <br>
 +
. Gibson Assembly mastermix <br>
 +
. Digest P<sub>bad</sub> Key dTT and P<sub>tet</sub> Lock 1 GFP dTT for 3A assembly <br>
 +
. Miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
 +
. 3A assembly <br>
 +
. Plan for P<sub>bad</sub> characterization <br>
 +
. Prepare stock arabinose solution M9 medium <br>
 +
 
 +
<br><b>Aug 14</b> <br>
 +
Wet lab<br>
 +
. Gel check for lock3.GFP.dTT colony PCR I-VI <br>
 +
. check dNTPs <br>
 +
. miniprep TA.dTTs <br>
 +
. restriction check TA.dTTs <br>
 +
. miniprep Lock3.GFP.dTT <br>
 +
. colony PCR for P<sub>bad</sub>-key3-dTT <br>
 +
. Gel extraction-purification of constitutive promoter <br>
 +
. Ligate CR.GFP.dTTs into constitutive promoter <br>
 +
. ligate TA.dTT with P<sub>bad</sub> <br>
 +
. transform ligate P<sub>bad</sub>.TA.dTTs <br>
 +
. transform constitutive promoter.CR.GFP.dTTs <br>
 +
 
 +
<br><b>Aug 15</b> <br>
 +
Wet lab<br>
 +
. Gibson Assembly mastermix <br>
 +
. purify CR.GFP.dTTs <br>
 +
. Ligate CR.GFP.dTTs into constitutive promoter <br>
 +
. Digestion of TA-dTT and PCR clean-up <br>
 +
. Restriction Check for TA-dTT <br>
 +
. transform ligate P<sub>bad</sub>.TA.dTTs <br>
 +
. transform constitutive promoter.CR.GFP.dTTs <br>
 +
. make 10x DNA loading Dye <br>
 +
. Order new TA and CR <br>
 +
. Gibson Assembly mastermix <br>
 +
 
 +
 
 +
<br><b>Aug 16</b> <br>
 +
Wet lab<br>
 +
. Miniprep <br>
 +
. Colony PCR for final constructs <br>
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 4</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 18</b> <br>
 +
Wet lab<br>
 +
. Restriction check for P<sub>bad</sub>.TA.dTTs <br>
 +
. Restriction check for constitutive promoter.CR.GFP.dTT <br>
 +
. Restriction check for constitutive promoter.RBS.GFP.dTT <br>
 +
. PCR for CR.GFP.dTT <br>
 +
. PCR for RBS.GFP.dTT <br>
 +
. PCR check for constitutive promoter.CR.GFP.dTT <br>
 +
. PCR check for constitutive promoter.RBS.GFP.dTT <br>
 +
. PCR check for P<sub>bad</sub>.TA.dTT <br>
 +
. restriction check TA.dTTs <br>
 +
. Prepare functional assay for RBS, CRs <br>
 +
. Inoculate candidates <br>
 +
. Prepare pSB3K3 <br>
 +
. Prepare pSB1C3 for oligo ligation <br>
 +
. Prepare BBa_I13401 for oligo ligation <br>
 +
. Prepare BBa_B0015 for oligo ligation <br>
 +
. Inoue competent cells <br>
 +
. Prepare for P<sub>bad</sub> characterization <br>
 +
. Take over the tank B and C around 1pm-5;30 <br>
 +
 
 +
<br><b>Aug 19</b> <br>
 +
Wet lab<br>
 +
. Gel check for O/NPCR products <br>
 +
. Gel check for PCR products (18/08) <br>
 +
. miniprep c.Promoter.Lock 1RBS.GFP.dTT <br>
 +
. miniprep possible candidates <br>
 +
. Restrict TA.dTT <br>
 +
. pSB1C3-BBa_I13401 for oligo ligation <br>
 +
. BBa_B0015 for oligo ligation <br>
 +
. pSB1C3-BBa_J04450 for oligo ligation <br>
 +
. Inoue competent cells <br>
 +
. P<sub>bad</sub>-key3-dTT restriction check <br>
 +
 
 +
<br><b>Aug 20</b> <br>
 +
Wet lab<br>
 +
. Gel check for P<sub>bad</sub>.TA.dTTs <br>
 +
. purification for BBa_B0015, BBa_I13401 <br>
 +
. pSB1C3-BBa_J04450 for oligo ligation <br>
 +
. Gel check for PCR products (19/08) <br>
 +
. PCR check for BBa_J23102, LOCK 3 GFP DTT <br>
 +
. Restriction check for key1 DTT <br>
 +
 
 +
<br><b>Aug 21</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for final constructs <br>
 +
. Ligate UST lock <br>
 +
. restriction check for key1.dTT <br>
 +
. ligate P<sub>bad</sub>.key1.dTT <br>
 +
. ligate lock 3.GFP.dTT with C. promoter <br>
 +
. Colony PCR for c. promoter.MCR.GFP.dTT <br>
 +
. competent cells <br>
 +
. Glycerol stocks <br>
 +
. transform HKUSTlock.GFP.dTT <br>
 +
. Transform pSB1C3-HKUSTlock <br>
 +
 
 +
 
 +
<br><b>Aug 22</b> <br>
 +
Wet lab<br>
 +
. competent cells <br>
 +
. Colony PCR for pSB1C3-HKUSTlock1 <br>
 +
. Colony PCR for HKUSTlock1.GFP.dTT <br>
 +
. miniprep pSB3K3-J04550 for pSB3K3 Backbone <br>
 +
. restrict pSB3K3 for 3 pieces assemblage <br>
 +
. ligate TA-CR into pSB3K3 <br>
 +
. Transform TA-CR <br>
 +
. colony PCR for P<sub>bad</sub>-key3-dTT <br>
 +
. inoculate P<sub>bad</sub>.key1.dTT <br>
 +
. gel check for C. PCR on 21/08 <br>
 +
. extraction of pSB3K3 <br>
 +
. inoculate HKUSTlock1.GFP.dTT <br>
 +
. inoculate pSB1C3-HKUSTlock1 <br>
 +
 
 +
 
 +
<br><b>Aug 23</b> <br>
 +
Wet lab<br>
 +
. Gel check for TAs <br>
 +
. colony PCR for UST lock in BBa_I13401 and pSB1C3 <br>
 +
. transform ust key <br>
 +
. Check competent cells <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 24</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for P<sub>bad</sub>.Key1.dTT <br>
 +
. Gel check for P<sub>bad</sub>.Key1.dTT <br>
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
<div>
 +
      <a href="#" class="head">Week 5</a>
 +
      <div class="content" align="left" style="display: none;">
 +
<br><b>Aug 25</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for P<sub>bad</sub>.Key1.dTT <br>
 +
. PCR check for CR.GFP.dTT <br>
 +
. PCR check for Cons. prom.CR.GFP.dTT <br>
 +
. anneal and ligate ust lock <br>
 +
. anneal and ligate ust key <br>
 +
. transform ust lock1 <br>
 +
. transform ust key <br>
 +
. ligation again key 1 into BBa_B0015 <br>
 +
 
 +
 
 +
<br><b>Aug 26</b> <br>
 +
Wet lab<br>
 +
. Colony PCR for "un-named lock" <br>
 +
. inoculate ones which SEEMS like it contains lock <br>
 +
. Colony PCR for "un-named key" <br>
 +
. inoculate ones which SEEMS like it contains key <br>
 +
. colony PCR for Key1.DTT <br>
 +
. Gel check for O/NPCR products <br>
 +
. PCR check for CR.GFP.dTTs <br>
 +
. Inoculate all possible candidates <br>
 +
. Prepare BBa_I13401 for CRs <br>
 +
. Streak possible c.p.CR/RBS.GFP.dTT <br>
 +
. make SOC <br>
 +
. single cut check for TAs <br>
 +
. make 5 alpha cell <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 27</b> <br>
 +
Wet lab<br>
 +
. miniprerp candidates <br>
 +
. PCR check for candidates <br>
 +
. gel check for o/n PCR <br>
 +
. restriction check for ust key 1 <br>
 +
. restriction check for key 1 <br>
 +
. P<sub>bad</sub> for ust key 1 <br>
 +
. extract ust lock 1 for BBa_J23102 <br>
 +
. ligate ust lock and key <br>
 +
. transform ust lock and key <br>
 +
. prepare plates for macroscope <br>
 +
. prepare dna for sequencing <br>
 +
. SOB <br>
 +
. SOC <br>
 +
. oligo annealing <br>
 +
. plasmid PCR for HKUSt lock and key <br>
 +
. overnight digestion of B0034 for control construct <br>
 +
. Digestion of HKUST key-BBa_B0015 <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 28</b> <br>
 +
Wet lab<br>
 +
. miniprep locks <br>
 +
. glycerol stocks for locks <br>
 +
. minipreps for BBa_B0015 and B0034 <br>
 +
. ligation of P<sub>bad</sub>-Hkust key-BBa_B0015 <br>
 +
. digestion of HKUST lock <br>
 +
. ligation of HKUST lock with GFP <br>
 +
. ligation of HKUST key and lock (4th) <br>
 +
. ligation of B0034 with GFP <br>
 +
. colony PCR of HKUST key and lock <br>
 +
. inoculation of key and lock <br>
 +
. miniprep <br>
 +
. Key one restriction check <br>
 +
. key one restriction for inserting P<sub>bad</sub> <br>
 +
. Restrict and extract lock3c rbs and ligate into c.promoter <br>
 +
. ligate lock 3c.GFP.dTT with C. promoter <br>
 +
. transform c. promoter lock 3c rbs.GFP.DTT <br>
 +
. Extract mcr/mta <br>
 +
. ligate MCR and MTA into pSB3K3 <br>
 +
. transform MCR/MTA <br>
 +
. Glycerol stock for locks <br>
 +
. BBa_E0240 ligate with C. Promoter <br>
 +
. TB <br>
 +
. parallel ligation and anealing for key 1 <br>
 +
. Sequencing preparations <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 29</b> <br>
 +
Wet lab<br>
 +
. minipreps <br>
 +
. Gel extraction, purification for O/N digestions <br>
 +
. competent cells <br>
 +
. Extract mcr/mta <br>
 +
. ligate MCR and MTA into pSB3K3 <br>
 +
. transform MCR/MTA <br>
 +
. Glycerol stock for locks <br>
 +
. PCR check for Biobrick key1 <br>
 +
 
 +
 
 +
 
 +
<br><b>Aug 30</b> <br>
 +
Wet lab<br>
 +
. inoculation of 12 falcons <br>
 +
. Colony PCR for pSB3K3 constructs <br>
 +
. Transform key1 O/N <br>
 +
. miniprep <br>
 +
. Competent cells <br>
 +
. AseI check Key 1 Dtt <br>
 +
 
 +
 
 +
 
 +
      </div>
 +
    </div>
 +
  </div>
 +
 
<!---- SSSSSSSS  EEEEEEEE  PPPPPPPP  TTTTTTTTTTTT  EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  RRRRRRRR    2 0 1 3-------->
<!---- SSSSSSSS  EEEEEEEE  PPPPPPPP  TTTTTTTTTTTT  EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  RRRRRRRR    2 0 1 3-------->
<!---- S          E          P      P      T        E          MM      MM  B      B  E          R    R 2 0 1 3-------->
<!---- S          E          P      P      T        E          MM      MM  B      B  E          R    R 2 0 1 3-------->
Line 732: Line 2,042:
<!---- SSSSSSSS  EEEEEEEE  P              T        EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  R  R 2 0 1 3-------->
<!---- SSSSSSSS  EEEEEEEE  P              T        EEEEEEEE  M        M  BBBBBBBB    EEEEEEEEE  R  R 2 0 1 3-------->
-
<div id="satu" align="center"> <h2>September 2013</h2>
+
<div id="satu" align="center"> <h2>September 2014</h2>
    
    
<div>
<div>
       <a href="#" class="head">Week 1</a>
       <a href="#" class="head">Week 1</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>August 2</b> <br>
+
<br><b>Sept 1</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. restriction check P<sub>bad</sub>- Lost your key-DTT <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. restriction check P<sub>bad</sub>-Lost your key-DTT <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. colony PCR for CR.TAs (orthogonality) <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. colony PCR for Key1,DTT <br>
-
<br><b>Sept xx</b> <br>
+
. ligate lock3c.GFP.dTT into c.p plasmid <br>
 +
. transform lock3.GFP.DTT <br>
 +
. Colony PCR for CR.TAs (orthogonality) <br>
 +
. Colony PCR for sherLock-GFP-DTT <br>
 +
. restriction check for Lost Your Key in pSB1C3 <br>
 +
. transform P<sub>bad</sub>.lost your key.DTT <br>
 +
. inoculate orthogonality candidates <br>
 +
. Prepare midi-prep for pSB3K3 <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 2</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. Gel check for O/N colony PCR for key1 and pSB3K3 <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. massive prep <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. PCR check sense reverse <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. colony PCR for orthogonality <br>
-
<br><b>Sept xx</b> <br>
+
. colony PCR for cons. pro. lock3crbs.GFP.DTT <br>
 +
. migrate cpromoter BBa_E0240 to pSB3K3 with mta key <br>
 +
. inoculation of B0034-GFP-DTT <br>
 +
. inoculation of Sherlock-GFP-DTT <br>
 +
. sequencing for sherlock in pSB1C3 <br>
 +
. digestion of B0034-GFP-DTT <br>
 +
. digestion of sherlock-GFP-DTT <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 3</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. digestion of P<sub>bad</sub> MTA dTT <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. digestion of P<sub>bad</sub> dTT <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. digestion of BBa_J23102 MCR GFP dTT <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. digestion of BBa_J23102 BBa_E0240(BBa_B0032 GFP dTT) <br>
-
<br><b>Sept xx</b> <br>
+
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450) <br>
 +
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
 +
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br>
 +
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> dTT <br>
 +
. Plasmid PCR for SherLock-GFP-DTT <br>
 +
. ligation of B0034-GFP-DTT <br>
 +
. ligation of SherLock-GFP-DTT <br>
 +
. sequencing <br>
 +
. check plate for arabinose induced mcr mta while singing <br>
 +
. extraction of key and lock <br>
 +
. restriction check for key with lock <br>
 +
. extraction of key1 DTT <br>
 +
. ligation of key1 DTT with P<sub>bad</sub> <br>
 +
. Gel check for O/N colonyPCR <br>
 +
. PCR check <br>
 +
. Check 031 <br>
 +
. ligate lock3c RBS.GFP.dTT into c.p plasmid <br>
 +
. transform lock3c RBS.GFP.dTT <br>
 +
. Midi-prep preparations <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 4</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. Gel purification of digested insert and backbone <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. digestion of BBa_J23102 BBa_E0240(BBa_B0032 GFP dTT) <br>
-
<br><b>Sept xx</b> <br>
+
. Transformation of ligated projects mentioned above <br>
 +
. Colony PCR for sherLock-GFP-DTT <br>
 +
. Colony PCR for B0034-GFP-DTT <br>
 +
. Inoculation of successful colonies <br>
 +
. Gel check for O/N Colony PCR <br>
 +
. Gel check for O/N PCR <br>
 +
. Gel check for Erics restriction check <br>
 +
. Inoculation of successful colonies <br>
 +
. Midi-prep preparations <br>
 +
. Gel purification <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 5</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. miniprep/ glycerol stock <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. Transform P<sub>bad</sub> dTT <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. Transform ligated produce of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
 
 +
 
 +
 
Line 774: Line 2,145:
       <a href="#" class="head">Week 2</a>
       <a href="#" class="head">Week 2</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Sept xx</b> <br>
+
<br><b>Sept 8</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      <br>
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·   <br>
-
<br><b>Sept xx</b> <br>
+
·      <br>
 +
·      <br>
 +
·      <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 9</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      <br>
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·   <br>
-
<br><b>Sept xx</b> <br>
+
·      <br>
 +
·      <br>
 +
 
 +
<br><b>Sept 10</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. colony PCR <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. ligations for final constructs <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. inoculations <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. P<sub>bad</sub>.lost your key.DTT <br>
-
<br><b>Sept xx</b> <br>
+
. sherlock <br>
 +
. gel check for colony PCR <br>
 +
. colony PCR <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 11</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      <br>
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·   <br>
-
<br><b>Sept xx</b> <br>
+
 
 +
<br><b>Sept 12</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. colony PCR <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. restrict key1.DTT for P<sub>bad</sub> insertion <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. ligate P<sub>bad</sub> with key1.DTT <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. transform key1.dTT <br>
 +
. extract pSB3K3 <br>
 +
. Prepare TB <br>
 +
. Prepare dextrose for SOC <br>
 +
. check sequencing results <br>
 +
. Digestion of CLON041 for constructs <br>
 +
. ligation <br>
 +
. inoculation <br>
 +
. Digestion of CLON 036 <br>
 +
. colony PCR mcr mta <br>
 +
 
 +
 
       </div>
       </div>
Line 810: Line 2,209:
       <a href="#" class="head">Week 3</a>
       <a href="#" class="head">Week 3</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Sept xx</b> <br>
+
<br><b>Sept 15</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
·      <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
·      <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
·      <br>
-
·     Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
·   <br>
-
<br><b>Sept xx</b> <br>
+
 
 +
 
 +
<br><b>Sept 16</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. mini prep P<sub>bad</sub>.key1.DTT <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. restriction check P<sub>bad</sub>.key1.DTT <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. gel check for O/N c.PCR <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. extract P<sub>bad</sub>.key1 <br>
-
<br><b>Sept xx</b> <br>
+
. inoculate every CR.TA construct <br>
 +
. ligate transform key 1 with locks <br>
 +
. miniprep (B1, B2, and B3) <br>
 +
. plasmid PCR B1, B2 and B3 using sense oligo and VR <br>
 +
. Overnight digestion for ones which give positive results <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 17</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. purify P<sub>bad</sub>.key1 <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. ligate P<sub>bad</sub>key1 DTT with locks <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. transform locks.P<sub>bad</sub>.key1.DTT <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. 2xYT (agar) AMP <br>
-
<br><b>Sept xx</b> <br>
+
. transform all <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 18</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. Spread a plate <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. miniprep for 3 inoculations <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. Sense oligo and VR PCR for HKUST Lock-DTT construct <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. Overnight digestion of plasmid which gives a band around 1300bp <br>
-
<br><b>Sept xx</b> <br>
+
. EtoHpp for sequencing <br>
 +
. Miniprep constructs for submission <br>
 +
. inoculation <br>
 +
. transform 031, 034 <br>
 +
. Check "Diagonal constructs" and controls <br>
 +
. migrate CR constructs into pSB3K3 <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 19</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. miniprep lock.key candidate <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. ligate problematic cr.key <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. restriction check sherlock <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. gel extraction<br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 20</b> <br>
 +
Wet lab<br>
 +
. miniprep CR.TA <br>
 +
. Colony PCR for CR.TA <br>
 +
. Colony PCR for CR.TA orthogonality <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 21</b> <br>
 +
Wet lab<br>
 +
. miniprep matching CR.TA <br>
 +
. Restriction check by EcoR1 and Pst1 <br>
 +
 
 +
 
Line 847: Line 2,289:
       <a href="#" class="head">Week 4</a>
       <a href="#" class="head">Week 4</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Sept xx</b> <br>
+
<br><b>Sept 22</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. Run Gel check for matching set of CR.TA <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. ligate problematic sets <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. transform problematic sets <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. prepare DNA for submission <br>
-
<br><b>Sept xx</b> <br>
+
. Gel purification <br>
 +
. Ligation lock-DTT with constitutive promoter <br>
 +
. transformation <br>
 +
. pSB3K3-CRs ligation <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 23</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. pSB3K3-CRs transformation <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. Gel extractions <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. Gel purifications <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. TB <br>
-
<br><b>Sept xx</b> <br>
+
. Agar L (+) plates <br>
 +
. Send DNA to BGI for sequencing <br>
 +
. C. PCR <br>
 +
. Inoculate possible candidates <br>
 +
. ligate problematic sets <br>
 +
. transform confirmed constructs <br>
 +
. inoculation for HKUST lock Construct <br>
 +
. colony PCR For lock <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 24</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. Competent cells preparations <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. minipreps <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. colony PCR <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. miniprep for Biobrick submission <br>
-
<br><b>Sept xx</b> <br>
+
. send plasmid for sequencing <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 25</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. insane prep <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. restriction check <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. Gel check for previous PvuII digestions <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
 
-
<br><b>Sept xx</b> <br>
+
 
 +
 
 +
 
 +
<br><b>Sept 26</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. Gel check PvuII digestions <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. Gel check AseI digestions <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. 3A assembly for non pSB1C3 <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. final batch of ligation for all missing component <br>
 +
. prepare DNA for submission <br>
 +
. Extraction for ligation <br>
 +
. Checking constructs-3 constructs using digestion check <br>
 +
. transformation for LOCK-8 <br>
 +
. Restriction check for controls for MCR/MTA <br>
 +
. Transformation of confirmed plasmid <br>
 +
 
 +
 
 +
<br><b>Sept 27</b> <br>
 +
Wet lab<br>
 +
. C. PCR for H series... <br>
 +
. inoculate possible candidates <br>
 +
. check self ligated pSB3K3 <br>
 +
. Gel check for possible candidates AseI <br>
 +
. Gel check for possible candidates PvuII <br>
 +
. Ligated H6 H7 <br>
 +
. transformed H6 H7 <br>
 +
. O/N inoculation of MCR/MTA for characterization <br>
 +
 
 +
 
 +
 
 +
<br><b>Sept 28</b> <br>
 +
Wet lab<br>
 +
. Extract pSB1C3 <br>
 +
. Extract 036 <br>
 +
. miniprep "H" series candidates <br>
 +
. Repeat CHL restrictions <br>
 +
. Repeat CHL ligations <br>
 +
. Repeat CHL transformations <br>
 +
. Gel check for "7" series using NcoI <br>
 +
. restriction check AseI <br>
 +
. restriction check NcoI <br>
 +
. Fluorescence measurement of MCR/MTA for characterization. <br>
 +
. Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn) <br>
 +
 
 +
 
 +
 
Line 884: Line 2,389:
       <a href="#" class="head">Week 5</a>
       <a href="#" class="head">Week 5</a>
       <div class="content" align="left" style="display: none;">
       <div class="content" align="left" style="display: none;">
-
<br><b>Sept xx</b> <br>
+
<br><b>Sept 29</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. restriction check AseI <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. restriction check NcoI <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. mini prep 6 + MCR/MTA (thanks from Doyle) <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. Restriction check for MCR/MTA in pSB3K3. <br>
-
<br><b>Sept xx</b> <br>
+
. restrict 036 <br>
 +
. purify 036 <br>
 +
. ligate monkey with sherlock <br>
 +
. Gel check for NcoI and AseI <br>
 +
. Get pSB1C3 backbone EcoRI and PstI <br>
 +
. Fluorescence measurement of MCR/MTA for characterization (second trial) <br>
 +
. inoculate Lock and Keys for crude functional assay. <br>
 +
 
 +
 
 +
 
 +
 
 +
<br><b>Sept 30</b> <br>
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. miniprep 11 tubes in shaker c <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. restriction check H" series with NcoI <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
. Restrict inoculated colonies wiht AseI <br>
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
. Restrict inoculated colonies wiht NcoI <br>
-
<br><b>Sept xx</b> <br>
+
. Gel purifications <br>
-
Wet lab<br>
+
. ligation of H series and some from other series... <br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. transformation of 16 confirmed constructs <br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
 
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
 
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
<br><b>Oct 1</b> <br>
-
<br><b>Sept xx</b> <br>
+
Wet lab<br>
Wet lab<br>
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
. digestion of key3,HKUSTkey<br>
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
. miniprep- final constructs <br>
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
 
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
-
<br><b>Sept xx</b> <br>
+
-
Wet lab<br>
+
-
·      Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF<br>
+
-
·      Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification<br>
+
-
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase<br>
+
-
·      Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol<br><br>
+
Line 919: Line 2,427:
     </div>
     </div>
<hr>
<hr>
-
</div>
 
</div>
</div>
</td>
</td>
</tr>
</tr>
</table>
</table>
-
<table class= "site_map_table" align= "center">
+
</div>
-
<tr>
+
</div>
-
<td class= 'site_map_column'><a href="#"><h4>Home</h4></a>
+
<footer>
-
<ul class= 'site_list'>
+
<script>
-
<li class='site_link'><a href="#">Team</li></a>
+
$(document).ready(function(){
-
<li class='site_link'><a href="#">Project</li></a>
+
-
<li class='site_link'><a href="#">WetLab</li></a>
+
//Check to see if the window is top if not then display button
-
<li class='site_link'><a href="#">Achievement</li></a>
+
$(window).scroll(function(){
-
<li class='site_link'><a href="#">Human Practice</li></a>
+
if ($(this).scrollTop() > 100) {
-
<li class='site_link'><a href="#">Side Project</li></a>
+
$('.scrollToTop').fadeIn();
-
</ul>
+
} else {
-
 
+
$('.scrollToTop').fadeOut();
-
</td>
+
}
-
<td class= 'site_map_column'><a href="#"><h4>Team</h4></a>
+
});
-
<ul class= 'site_list'>
+
-
<li class='site_link'><a href="#">Member</li></a>
+
//Click event to scroll to top
-
<li class='site_link'><a href="#">Advisers</li></a>
+
$('.scrollToTop').click(function(){
-
<li class='site_link'><a href="#">Instructor</li></a>
+
$('html, body').animate({scrollTop : 0},800);
-
<li class='site_link'><a href="#">Attribution</li></a>
+
return false;
-
<li class='site_link'><a href="#">Acknowledgement</li></a>
+
});
-
</ul>
+
-
</td>
+
});
-
<td class= 'site_map_column'><a href="#"><h4>Main Project</h4></a>
+
</script>
-
<ul class= 'site_list'>
+
<a href="#" class="scrollToTop">
-
<li class='site_link'><a href="#">Overview</li></a>
+
<div class="scrollToTop">
-
<li class='site_link'><a href="#">Module one</li></a>
+
<br>
-
<li class='site_link'><a href="#">Module two</li></a>
+
Back to top
-
<li class='site_link'><a href="#">Data</li></a>
+
</div>
-
<li class='site_link'><a href="#">Characterisation</li></a>
+
</a>
-
</ul>
+
</footer>
-
</td>
+
-
<td class= 'site_map_column'><a href="#"><h4>Side Project</h4></a>
+
-
<ul class= 'site_list'>
+
-
<li class='site_link'><a href="#">Overview</li></a>
+
-
<li class='site_link'><a href="#">Parts</li></a>
+
-
<li class='site_link'><a href="#">Catalogue</li></a>
+
-
<li class='site_link'><a href="#">Feature Pages</li></a>
+
-
</ul>
+
-
</td>
+
-
<td class= 'site_map_column'><a href="#"><h4>Human Practice</h4></a>
+
-
<ul class= 'site_list'>
+
-
<li class='site_link'><a href="#">Overview</li></a>
+
-
<li class='site_link'><a href="#">Start up kit</li></a>
+
-
<ul>
+
-
<li class='site_link'><a href="#">Interview</li></a>
+
-
<li class='site_link'><a href="#">Report</li></a>
+
-
<li class='site_link'><a href="#">Database</li></a>
+
-
</ul>
+
-
<li class='site_link'><a href="#">Talks</li></a>
+
-
<li class='site_link'><a href="#">Outreach</li></a>
+
-
</ul>
+
-
</td>
+
-
<td class= 'site_map_column'><a href="#"><h4>Wetlab</h4></a>
+
-
<ul class= 'site_list'>
+
-
<li class='site_link'><a href="#">Protocol</li></a>
+
-
<li class='site_link'><a href="#">Notebook</li></a>
+
-
<li class='site_link'><a href="#">Safety</li></a>
+
-
</ul>
+
-
</td>
+
-
<td class= 'site_map_column'><a href="#"><h4>Achievement</h4></a>
+
-
<ul class= 'site_list'>
+
-
<li class='site_link'><a href="#">Medal Requirements</li></a>
+
-
<li class='site_link'><a href="#">Deliverable</li></a>
+
-
</ul>
+
-
</td>
+
-
</tr>
+
-
 
+
-
</table>
+
-
 
+
-
+
-
+
</body>
</body>
 +
</html>
</html>
 +
}}

Latest revision as of 00:38, 18 October 2014



Notebook

Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and research findings. The notebook for Project Pneuumonsensor and Project Riboregulator are displayed separately.






Pneumosensor

June 2014

Week 1
Week 2
Week 3
Week 4

July 2014

Week 1
Week 2
Week 3
Week 4
Week 5

August 2014

Week 1
Week 2
Week 3
Week 4
Week 5

September 2014

Week 1
Week 2
Week 3
Week 4
Week 5





Riboregulator

July 2014

Week 1
Week 2
Week 3
Week 4

August 2014

Week 1
Week 2
Week 3
Week 4
Week 5

September 2014

Week 1
Week 2
Week 3
Week 4
Week 5


Back to top

Home

Pneumosensor

Riboregulator

Human Practice

Team

WetLab

Achievement