Team:Hong Kong HKUST/notebook/mod2

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<body>
<body>
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          <a href="https://2013.igem.org/Main_Page"><img id="iGEM_Logo" src="https://static.igem.org/mediawiki/2013/4/46/Igem_qgem_logo.png"></a>
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<a href="http://www.ust.hk/eng/index.htm"><img id="hkust_Logo" src="https://static.igem.org/mediawiki/2013/5/55/Hkust_logo.gif"></a>
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<ol class="pos_fixed">
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod1">Module 1</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod2">Module 2</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod3">Module 3</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod4">Module 4</a></il>
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</ol>
<a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a>
<a href=https://2013.igem.org/Team:Hong_Kong_HKUST><center><div id="kepala" style="height:121px;width:100%;"><img src="https://static.igem.org/mediawiki/igem.org/c/c7/BANNER1_%281%29.png" style="height:121px;width:100%;align:middle;"></div></center></a>
<div id="cover"></div>
<div id="cover"></div>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/advisors">Advisors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/instructors">Instructors</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/attribution">Attribution</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/acknowledge">Acknowledgement</a></li>
</ul>
</ul>
</li>
</li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/project">Project</a>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Project">Project</a>
<ul>
<ul>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/abstract">Abstract</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modelling">Modelling</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modules">Modules Description</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/data">Data Page</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Parts">Parts</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/datapage">Data Page</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/characterization">Characterization</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Results</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/results">Result</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/future">Future Work</a></li>
</ul>
</ul>
</li>
</li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/Wetlab">Wetlab</a>
<ul>
<ul>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">notebook</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/notebook">Notebook</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">protocols</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/protocols">Protocols</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">safety</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">Safety</a></li>
 +
 
</ul>
</ul>
</li>
</li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp">Human Practice</a>
<ul>
<ul>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Interviews">Interviews</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/cp">Country Profile</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Presentation">Presentation</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/blog">Blog</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Videos">Videos</a></li>
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<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/interview">Interviews</a></li>
-
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/Article">Article</a></li>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/article/genet">Article</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/video">Videos</a></li>
 +
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/hp/presentation">Presentations</a></li>
</ul>
</ul>
</li>
</li>
</ul>
</ul>
 +
</div>
</div>
-
<br><br><br><br><br><br><br>
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<br><br><br><br><br><br><br><br>
 +
<div id="title"><center><h3>FA Sensing Mechanism Module's Notebook</h3></center></div>
<div id="flight">
<div id="flight">
  <div id="satu" align="center"> <h1>June 2013</h1>
  <div id="satu" align="center"> <h1>June 2013</h1>
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       <div>
       <div>
       <a href='#' class='head'>Week 4</a>
       <a href='#' class='head'>Week 4</a>
-
       <div class='content' align="left">Wet lab<br>
+
       <div class='content' align="left">
-
·      Inoculation of pBlueScript KS(+) for training<br>
+
<br><b>June 24</b><br>
-
·      Autoclave basic materials<Br>
+
Wet lab <br>
-
·      Preparing LB<br>
+
·      Inoculation of pBlueScript KS(+) for training <br>
-
·      LB-Ampicillin plates poured<br>
+
·      Autoclave basic materials <br>
-
Dry lab<br>
+
·      Preparing LB <br>
-
Protocols review
+
·      LB-Ampicillin plates poured
 +
Dry lab <br>
 +
Protocols review <br>
 +
 
 +
<br><b>
 +
June 25 </b><br>
 +
Wet lab <br>
 +
·  Plasmid extraction of pBlueScript KS(+)  <br>
 +
·      LB-Chloramphenicol plates poured <br>
 +
Dry lab <br>
 +
·      Primers design for FABP1 promoter, PPAR-alpha promoter, GRP78 promoter <br>
 +
·      Informal meeting <br>
 +
 
 +
<br><b>
 +
June 26 </b><br>
 +
Wet lab <br>
 +
·      Extraction of gDNA from HepG2 Cells for FABP1 promoter and PPAR-alpha promoter <br>
 +
·      Transformation of pEGFP-N1 and <a href="http://parts.igem.org/Part:BBa_K817002">BBa_K817002</a> (P<i>fadBA</i>) <br>
 +
 
 +
<br><b>
 +
June 27 </b><br>
 +
Wet lab <br>
 +
·      Inoculation of pEGFP-N1 and BBa_K817002 (P<i>fadBA</i>) for plasmid extraction <br>
 +
 
 +
<br><b>
 +
June 28</b> <br>
 +
Wet lab <br>
 +
·      Extraction of pEGFP-N1 and BBa_K817002 (P<i>fadBA</i>)  plasmids by miniprep <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>
 +
·      Restriction of c(P<i>fadBA</i>) by EcoR1 and Pst1 <br>
 +
·      Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (P<i>fadBA</i>) <br>
 +
 
 +
<br><b>
 +
June 30 </b><br>
 +
Dry lab <br>
 +
Experiment planning and protocols revision for next week work <br><br>
 +
 
       </div>
       </div>
       </div>
       </div>
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   <div>
   <div>
       <a href='#' class='head'>Week 1</a>
       <a href='#' class='head'>Week 1</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<br> <b> July 2 </b> <br>
 +
Wet lab <br>
 +
·      Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter <br>
 +
·      LB-Chloramphenicol plates (the previous ones were found contaminated) <br>
 +
·      Inoculation of pEGFP-N1 for plasmid extraction <br>
 +
·      Transformation of P<i>fadBA</i> due chloramphenicol plates contamination <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>
 +
Wet lab<br>
 +
·      Inoculation of BBa_K817002  P<i>fadBA</i><br>
 +
·      PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<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>
 +
 
 +
<br> <b> July 5</b> <br>
 +
Wet lab<br>
 +
·      Extraction of BBa_K817002 (P<i>fadBA</i>) <br>
 +
·      Inoculation of pEGFP-N1<br>
 +
·      PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
 +
·      Gel check, 0.8% gel for previously gDNA extraction<br>
 +
·      Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter<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>
   </div>
   </div>
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   <div>
   <div>
       <a href='#' class='head'>Week 2</a>
       <a href='#' class='head'>Week 2</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<br> <b> July 8</b> <br>
 +
Wet lab<br>
 +
·      gDNA extraction from HepG2 cells<br>
 +
·      Restriction of BBa_K817002 P<i>fadBA</i><br>
 +
·      Gel check for gDNA extraction<br>
 +
·      Inoculation of BBa_J176171, BBa_K817002 (P<i>fadBA</i>), pEGFP-N1 for plasmid extraction<br>
 +
·      New primers for PPAR-alpha promoter and FABP1 promoter arrival<br>
 +
·      Ran gel for BBa_K817002 P<i>fadBA</i> restriction check, gel extraction and purification<br>
 +
 
 +
<br> <b> July 9</b> <br>
 +
Wet lab<br>
 +
·      Extraction of BBa_J176171, BBa_K817002 P<i>fadBA</i> and pEGFP-N1 by miniprep<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>
 +
·      Restriction of BBa_J176171 for P<i>fadBA</i> Ase1 and BamH1; FABP1 promoter by Xba1 and Not1<br>
 +
·      Ran gel for pEGFP-N1 and BBa_J176171 restriction products<br>
 +
·      PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA<br>
 +
·      <a href="http://parts.igem.org/Part:BBa_J52034">BBa_J52034</a> (<i>fad</i>R) transformation<br>
 +
 
 +
<br> <b> July 10</b> <br>
 +
Wet lab<br>
 +
·      Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter<br>
 +
·      PCR for FABP1 promoter, PCR replication<br>
 +
·      BBa_J176171 vector dephosphorylation  by antartic phosphatase<br>
 +
·      Ligation of BBa_K817002 and P<i>fadBA</i> and BBa_J176171 <br>
 +
·      BBa_J52034 (<i>fad</i>R) inoculation<br>
 +
·      Digestion for BBa_K817002 P<i>fadBA</i> extraction<br>
 +
Gel check for BBa_K817002 P<i>fadBA</i> extraction<br>
 +
 
 +
<br> <b> July 11</b> <br>
 +
Wet lab<br>
 +
·      Gel purification for BBa_K817002 P<i>fadBA</i> extraction<br>
 +
·      Gel check for FABP1 promoter PCR product<br>
 +
·      PCR clean-up<br>
 +
·      BBa_J52034 (<i>fad</i>R) miniprep<br>
 +
·      BBa_J52034 restriction by Pst1 HF and Not1 HF<br>
 +
·      PCR  for PPAR-alpha promoter<br>
 +
Dry lab<br>
 +
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions<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>
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   <div>
   <div>
       <a href='#' class='head'>Week 3</a>
       <a href='#' class='head'>Week 3</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<br><b>July 15</b> <br>
 +
Wet lab<br>
 +
·      Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation<br>
 +
·      Transformation of  FABP1 promoter, EGFP, and BBa_J176171 ligation<br>
 +
·      FABP1 promoter+EGFP +BBa_J176171 ligation restriction check<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>
 +
Wet lab<br>
 +
·      Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep<br>
 +
·      Repeat <i>fad</i>R and P<i>fadBA</i> constructs<br>
 +
·      Digest pEGFP-n1 and BBa-J176171 for <i>fad</i>R<br>
 +
·      Digestion for BBa_K817002 (P<i>fadBA</i>) extraction<br>
 +
·      Gel check and gel extraction for BBa_J176171 for <i>fad</i>R and BBa_K817002 (P<i>fadBA</i>) <br>
 +
 
 +
<br><b>July 18</b> <br>
 +
Wet lab<br>
 +
·      Gel purification for all digested products<br>
 +
·      FABP1 promoter digestion check for whole construct prior transfection<br>
 +
·      P<i>fadBA</i> vector de phosphorylation and ligation with EGFP and BBa_J176171<br>
 +
·      Transformation of P<i>fadBA</i>+EGFP+BBa_J176171<br>
 +
 
 +
<br><b>July 19</b> <br>
 +
Wet lab<br>
 +
·      FABP1 promoter preparation for transfection<br>
 +
·      Ligation of P<i>fadBA</i> and BBa_J176171 followed by transformation<br>
 +
·      PCR for PPAR-alpha promoter cloning<br>
 +
Dry lab<br>
 +
·      Design for characterization of the transfected cells with FABP1 construct<br>
 +
·      Review for Multiple Sites Mutagenesis<br>
 +
·      Ordered primers for pCMV cloning from pEGFP-N1<br><br>
 +
 
       </div>
       </div>
   </div>
   </div>
Line 334: Line 562:
   <div>
   <div>
       <a href='#' class='head'>Week 4</a>
       <a href='#' class='head'>Week 4</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<br><b>July 22</b> <br>
 +
Wet lab<br>
 +
·  FABP1 construct given for characterization and transfection<br>
 +
·  Further colony screening for FABP1 construct, inoculations<br>
 +
·  Inoculation of P<i>fadBA</i> and BBa_J176171<br>
 +
·  Ran gel for PPAR-alpha promoter PCR products<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>
 +
Wet lab<br>
 +
·      DNA purification from gel extraction for digested pEGFP-N1<br>
 +
·      Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter<br>
 +
·      Ran gel for pCMV cloning from pEGFP-N1<br>
 +
·      PCR clean up for pCMV cloning from pEGFP-N1<br>
 +
·      PCR for PPAR-alpha promoter with new primers using Taq polymerase<br>
 +
·      Transformation of mutagenesis products<br>
 +
·      Inoculation of P<i>fadBA</i> ligation colonies<br>
 +
 
 +
<br><b>July 25</b> <br>
 +
Wet lab<br>
 +
·      Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase<br>
 +
·      Minprep of P<i>fadBA</i>+EGFP+BBa_J176171 ligation colonies, followed by digestion check <br>
 +
 
 +
<br><b>July 26</b> <br>
 +
Wet lab<br>
 +
·      Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter<br>
 +
·      Ran gel before parental string digestion of mutagenesis products<br>
 +
·      Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check<br>
 +
·      Plasmids preparation according to transfection requirements, construct and controls<br><br>
 +
 
       </div>
       </div>
   </div>  
   </div>  
Line 340: Line 605:
   <div>
   <div>
       <a href='#' class='head'>Week 5</a>
       <a href='#' class='head'>Week 5</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<br><b>July 30</b> <br>
 +
Wet lab<br>
 +
·      PPAR-alpha promoter PCR cloning with new primers using Vent polymerase<br>
 +
·      Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1<br>
 +
·      BBa_J176171 vector de phosphorylation, followed by ligation with EGFP using T4 Ligase, then transformation into E. Coli strain DH10b<br>
 +
·      PCR for PPAR-alpha promoter with new primers using Vent polymerase<br>
 +
Dry lab<br>
 +
·      Discussion and re assessment of constructs related to pEGFP-N1<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>
 +
·      PCR for PPAR-alpha promoter with reference primers using Vent polymerase<br>
 +
·      Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase<br><br>
 +
 
       </div>
       </div>
   </div>
   </div>
Line 349: Line 630:
<div>
<div>
       <a href='#' class='head'>Week 1</a>
       <a href='#' class='head'>Week 1</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<br><b>August 2</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>
     </div>
     </div>
     <div>
     <div>
       <a href='#' class='head'>Week 2</a>
       <a href='#' class='head'>Week 2</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<br><b>August 5</b> <br>
 +
Wet lab<br>
 +
·      Gel Check for PPAR-alpha promoter PCR promoter cloning, using temperature gradient and every available set of primers designed. <br>
 +
·      Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with <i>fad</i>R followed by transformation<br>
 +
·      Ligation check of P<i>fadBA</i>+BBa_J176171+EGFP using Age1<br>
 +
·      PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase<br>
 +
·      gDNA Gel check<br>
 +
Dry lab<br>
 +
·      Primers design for <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>-PSB1C3 Xba1 restriction site removal by site direct mutagenesis<br>
 +
 
 +
<br><b>August 6</b> <br>
 +
Wet lab<br>
 +
·      Ran gel for PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase<br>
 +
·      Inoculation of <i>fad</i>R+BBa_J176171<br>
 +
 
 +
<br><b>August 7</b> <br>
 +
Wet lab<br>
 +
·      Digestion check of BBa_J176171+EGFP by Not1 and Pst1<br>
 +
·      Digestion check of BBa_J176171+<i>fad</i>R by Xba1 and HindIII<br>
 +
·      Ran gel for digestion check<br>
 +
·      Inoculation of <i>fad</i>R+BBa_J176171, colony screening<br>
 +
 
 +
<br><b>August 8</b> <br>
 +
Wet lab<br>
 +
·      Miniprep for <i>fad</i>R+BBa_J176171, colony screening<br>
 +
·      Digestion check of BBa_J176171+<i>fad</i>R by Xba1 and HindIII<br>
 +
·      Ran gel for digestion check<br>
 +
·      Inoculation of <i>fad</i>R+BBa_J176171, colony screening<br>
 +
·      pCMV from cloned from pEGFP-N1adding restriction sites for ligation with EGFP and BBa_J176171, followed by transformation<br>
 +
 
 +
<br><b>August 9</b> <br>
 +
Wet lab<br>
 +
·      Primers arrival for BBa_J04450-PSB1C3 for site direct mutagenesis<br>
 +
·      Digestion check of BBa_J176171+<i>fad</i>R Xba1 and HindIII, Xba1 and Spe1<br>
 +
·      Ran gel for digestion check<br>
 +
·      Site direct Mutagenesis for BBa_J04450-PSB1C3 removing Xba1 restriction site, followed by gel check before parental string digestion and transformation<br>
 +
·      Inoculation of pCMV from cloned from pEGFP-N1adding restriction sites for ligation with EGFP and BBa_J176171<br><br>
       </div>
       </div>
     </div>
     </div>
 +
     <div>
     <div>
       <a href='#' class='head'>Week 3</a>
       <a href='#' class='head'>Week 3</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p><strong>August 12 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep of pCMV+EGFP+ BBa_J176171, followed by digestion check and gel <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR for pCMV cloning from pEGFP-N1 using Vent polymerase, followed by gel check and PCR clean up <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion check of BBa_J176171+<i>fad</i>R by Xba1, HindIII and Spe1 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ran gel for digestion check <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;pEGFP-N1 digestion for GRP78, extraction pCMV using Ase1 and Xho1, followed by gel check and DNA purification <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with <i>fad</i>R followed by transformation <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site direct mutagenesis for BBa_J04450-PSB1C3, followed by gel check before parental digestion with Dpn1 and transformation</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 13 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Primers phosphorylation for FABP1 promoter Multi-site Mutagenesis using T4 PNK <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Primers phosphorylation for Site Direct Mutagenesis for BBa_J04450-PSB1C3 using T4 PNK <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis for BBa_J04450-PSB1C3 using phosphorylated primers and non phosphorylated primers <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Overnight culture for Competent cells <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;pDRIVE_hGRP78 arrival, transformation into <em>E. Coli</em> strain DH10b &nbsp;</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 14 </strong><br />
 +
  Wet lab <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Nothing done due the typhoon</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 15 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;P<i>fadBA</i>+pEGFP-N1 ligation, first vector dephosphorylation using Antarctic phosphatase followed by ligation by T4Ligase and transformation into <em>E. Coli</em> SURE strain. <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;pCMV+EGFP+BBa_J176171 digestion check, followed by gel check <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Repeat GRP78+pEGFP-N1 ligation, followed by transformation into <em>E. Coli</em> DH10b strain. <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion check for <i>fad</i>R+BBa_J176171 usign Xba1 and HindIII <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculations of <i>fad</i>R+ BBa_J176171, pCMV+EGFP+ BBa_J176171, P<i>fadBA</i>+pEGFP-N1, P<i>fadBA</i>+EGFP+ BBa_J176171 followed by gel check</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 16 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep of <i>fad</i>R+ BBa_J176171, pCMV+EGFP+ BBa_J176171, P<i>fadBA</i>+pEGFP-N1, P<i>fadBA</i>+EGFP+ BBa_J176171 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Restriction check for <i>fad</i>R+ BBa_J176171 and P<i>fadBA</i>+pEGFP-N1 followed by gel check <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculations of <i>fad</i>R+ BBa_J176171, P<i>fadBA</i>+pEGFP-N1, P<i>fadBA</i>+EGFP+ BBa_J176171 <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DH105alpha <em>E. Coli</em> competent cells preparation</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
     <div>
     <div>
       <a href='#' class='head'>Week 4</a>
       <a href='#' class='head'>Week 4</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p><strong>August 19 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Repeat GRP78+pEGFP-N1 ligation, followed by transformation into <em>E. Coli</em> DH10b strain. <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep of <i>fad</i>R+ BBa_J176171, P<i>fadBA</i>+pEGFP-N1, P<i>fadBA</i>+EGFP+ BBa_J176171 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Restriction check for P<i>fadBA</i>+pEGFP-N1 followed by gel check <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Restriction check for <i>fad</i>R+ BBa_J176171 and P<i>fadBA</i>+pEGFP-N1 followed by gel check <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Streak colony with the right <i>fad</i>R+BBa_J176171 construct <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DH5alpha <em>E. Coli </em>competent cells preparation <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br />
 +
</p>
 +
<p><strong>August 20</strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion check P<i>fadBA</i>+pEGFP-N1 with Pst1, Xba1. Followed by gel check <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion check for damaged plates of GRP78+pEGFP-N1 using Xba1 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion check of P<i>fadBA</i>+EGFP+ BBa_J176171 using Age1HF, HindIII and Xba1 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis for illegal restriction sites for FABP1 promoter<br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculate GRP78+pEGFP-N1, pCMV+EGFP+BBa_J176171 <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DH10b <em>E. Coli</em> competent cells preparation</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 21 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep GRP78+pEGFP-N1, followed by digestion check and gel check using Spe1 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep pCMV+EGFP+BBa_J176171 followed by digestion check using Nde1 and Age1HF <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion check of P<i>fadBA</i>+EGFP+BBa_J176171 using Nde1 , <i>fad</i>R+ BBa_J176171 using Age1 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site direct Mutagenesis for FABP1 promoter+EGFP+BBa_J176171 <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR for GRP78 promoter cloning from pDRIVE_hGRP78, followed by gel check PCR clean up, digestion using Ase1 and Xho1 and DNA purification</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 22 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DH10b <em>E. Coli</em> competent cells preparation <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site direct Mutagenesis for FABP1 promoter+EGFP+BBa_J176171 with phosphorylated primers, followed by gel check before Dpn1 parental string digestion <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligation of GRP78 and pEGFP-N1, first vector dephophorylation and ligation with T4Ligase. Transformed into <em>E. Coli</em> DH10b strain <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transformation of pBlueScript KS (+) in to SURE <em>E. Coli</em> <br />
 +
  Dry lab <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Discuss about FABP1 construct vanishing under PCR conditions</p>
 +
<p>&nbsp;</p>
 +
<div> </div>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
    
    
 +
<div>
 +
      <a href='#' class='head'>Week 5</a>
 +
      <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p><strong>August 26 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;FABP1 promoter assessment under PCR conditions <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;BBa_J176171+EGFP assessment under PCR conditions <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;BBa_J176171 assessment under PCR conditions <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Temperature gradient for FABP1 promoter+BBa_J176171+EGFP construct <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculate pBlueScript KS (+)</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 27 </strong><br />
 +
  Wet lab <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DH10b <em>E. Coli</em> competent cells preparation</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 28 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;BBa_J176171 assessment under PCR conditions <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Temperature gradient for FABP1 promoter+BBa_J176171+EGFP construct <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR for FABP1 promoter cloning from gDNA <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into <em>E.Coli</em> DH10b <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep pBlueScript KS (+) <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest pBlueScript KS (+) with Xba1 and BamH1HF <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Restreak BBa_J176171 and inoculate it</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 29 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep estreaked BBa_J176171 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into <em>E.Coli</em> DH10b <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ligation of FABP1 promoter in to pBlueScript KS (+) using T4 Ligase followed by transformation into DH10b <em>E. Coli</em> <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transformation of BBa_J176171 into SURE <em>E. Coli</em></p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 30 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculate GRP78+pEGFP-N1 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculate FABP1 promoter+pBlueScript KS (+) <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculate BBa_J176171 <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR conditions check for BBa_J176171 looking for heat degradation</p>
 +
<p>&nbsp;</p>
 +
<p><strong>August 31 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep of FABP1 promoter+pBlueScript KS (+), BBa_J176171 <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR conditions check for BBa_J176171 looking for heat degradation</p><br>
 +
      </div>
 +
    </div>
 +
  </div><div id="satu" align="center"> <h1>September 2013</h1>
  </div><div id="satu" align="center"> <h1>September 2013</h1>
    
    
     <div>
     <div>
       <a href='#' class='head'>Week 1</a>
       <a href='#' class='head'>Week 1</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p><strong>September 1 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion check for FABP1 promoter+pBlueScript KS (+) using EcoR1. Followed by gel check.</p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 2 </strong><br />
 +
  Wet lab <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digestion check for FABP1 promoter+pBlueScript KS (+) using EcoR1. Followed by gel check.</p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 3</strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Restriction check from the damaged plates GRP78+pEGFP-N1 using Xba1 <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Repeat FABP1 promoter and pBlueScript KS (+) ligation, vector Dephosphorylation by Antartic Phosphatase and ligation using T4 Ligase, followed by transformation into DH10b<em> E. Coli</em></p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 4 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GRP78 promoter PCR Cloning from pDRIVE_hGFRP78, followed by PCR cleanup using kit. Then restriction check using Xba1 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;pEGFP-N1 restriction for GRP78 assembly, digested by Ase1 and BamH1, then Gel check, Gel extraction and purification, vector dephosphorilation and ligation by T4 ligase. Transformed into DH10b <em>E. Coli</em> <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculation of FABP1 promoter+ pBlueScript KS (+)</p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 5 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep of FABP1 promoter+pBlueScript KS (+) then restriction check using Ecor1, followed by gel check. <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis for FABP1 promoter+pBlueScript KS (+), Gel Check</p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 6 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Restriction of GRP78 PCR product by Ase1 and Xho1 <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR for PPAR-alpha promoter cloning <br />
 +
  Dry lab <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Review on previous Mutagenesis attempts and troubleshooting</p>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
 +
     <div>
     <div>
       <a href='#' class='head'>Week 2</a>
       <a href='#' class='head'>Week 2</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p><strong>September 9 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Ran gel for PPAR-alpha promoter cloning <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;pEGFP-N1 &nbsp;digestion for PPAR-alpha promoter and vector dephosphorylation</p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 10 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis FABP1 promoter+pBlueScript KS(+) then Gel Check</p>
 +
<p>&nbsp; </p>
 +
<p><strong>September 11 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis FABP1 promoter+pBlueScript KS(+) then Gel Check <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Digest mutant FABP1 promoter+ pBlueScript KS(+) with Dpn1 then transformed into SURE <em>E. Coli</em></p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 12 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GRP78 PCR product digested by Ase1 and Xho1, ligated with dephosphorylated pEGFP-N1 vector and ligated by T4 ligase, transformed into DH10b<em> E. Coli </em> <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Inoculations of mutant FABP1 promoter+pBlueScript KS(+)</p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 13 </strong><br />
 +
  Wet lab <br />
 +
  · &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Miniprep of Mutant FABP1 promoter+ pBlueScript KS(+), then restriction check using Ecor1, ran gel <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check </p>
 +
<p>&nbsp;</p>
 +
<p><strong>September 14 </strong><br />
 +
  Wet lab <br />
 +
· &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check </p>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>
 +
     <div>
     <div>
       <a href='#' class='head'>Week 3</a>
       <a href='#' class='head'>Week 3</a>
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       </div>
       </div>
     </div>
     </div>
 +
     <div>
     <div>
       <a href='#' class='head'>Week 4</a>
       <a href='#' class='head'>Week 4</a>
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       </div>
       </div>
     </div>
     </div>
 +
     <div>
     <div>
       <a href='#' class='head'>Week 5</a>
       <a href='#' class='head'>Week 5</a>

Latest revision as of 23:14, 27 September 2013

  1. Notebook
  2. Module 1
  3. Module 2
  4. Module 3
  5. Module 4








FA Sensing Mechanism Module's Notebook

June 2013

Week 4

June 24
Wet lab
· Inoculation of pBlueScript KS(+) for training
· Autoclave basic materials
· Preparing LB
· LB-Ampicillin plates poured Dry lab
Protocols review

June 25
Wet lab
· Plasmid extraction of pBlueScript KS(+)
· LB-Chloramphenicol plates poured
Dry lab
· Primers design for FABP1 promoter, PPAR-alpha promoter, GRP78 promoter
· Informal meeting

June 26
Wet lab
· Extraction of gDNA from HepG2 Cells for FABP1 promoter and PPAR-alpha promoter
· Transformation of pEGFP-N1 and BBa_K817002 (PfadBA)

June 27
Wet lab
· Inoculation of pEGFP-N1 and BBa_K817002 (PfadBA) for plasmid extraction

June 28
Wet lab
· Extraction of pEGFP-N1 and BBa_K817002 (PfadBA) plasmids by miniprep
· 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
· Restriction of c(PfadBA) by EcoR1 and Pst1
· Ran 0.8% gel for pEGFP-N1 and 2% gel for BBa_K817002 (PfadBA)

June 30
Dry lab
Experiment planning and protocols revision for next week work

July 2013

Week 1

July 2
Wet lab
· Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter
· LB-Chloramphenicol plates (the previous ones were found contaminated)
· Inoculation of pEGFP-N1 for plasmid extraction
· Transformation of PfadBA due chloramphenicol plates contamination

July 3
· Extraction of pEGFP-N1 plasmids by miniprep
· PCR for PPAR-alpha promoter amplification
· Digestion of pEGFP-N1 by Ase1 and BamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter

July 4
Wet lab
· Inoculation of BBa_K817002 PfadBA
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· 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

July 5
Wet lab
· Extraction of BBa_K817002 (PfadBA)
· Inoculation of pEGFP-N1
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Gel check, 0.8% gel for previously gDNA extraction
· Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter
· Poured new LB-Kanamycin plates
· Transformation of BBa_J176171
Dry lab
· Discussion about re-design constructions, possible change for BBa_J176171 as mammalian expression vector
· Primer redesign for PPAR-alpha promoter and FABP1 promoter

Week 2

July 8
Wet lab
· gDNA extraction from HepG2 cells
· Restriction of BBa_K817002 PfadBA
· Gel check for gDNA extraction
· Inoculation of BBa_J176171, BBa_K817002 (PfadBA), pEGFP-N1 for plasmid extraction
· New primers for PPAR-alpha promoter and FABP1 promoter arrival
· Ran gel for BBa_K817002 PfadBA restriction check, gel extraction and purification

July 9
Wet lab
· Extraction of BBa_J176171, BBa_K817002 PfadBA and pEGFP-N1 by miniprep
· 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
· Restriction of BBa_J176171 for PfadBA Ase1 and BamH1; FABP1 promoter by Xba1 and Not1
· Ran gel for pEGFP-N1 and BBa_J176171 restriction products
· PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· BBa_J52034 (fadR) transformation

July 10
Wet lab
· Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter
· PCR for FABP1 promoter, PCR replication
· BBa_J176171 vector dephosphorylation by antartic phosphatase
· Ligation of BBa_K817002 and PfadBA and BBa_J176171
· BBa_J52034 (fadR) inoculation
· Digestion for BBa_K817002 PfadBA extraction
Gel check for BBa_K817002 PfadBA extraction

July 11
Wet lab
· Gel purification for BBa_K817002 PfadBA extraction
· Gel check for FABP1 promoter PCR product
· PCR clean-up
· BBa_J52034 (fadR) miniprep
· BBa_J52034 restriction by Pst1 HF and Not1 HF
· PCR for PPAR-alpha promoter
Dry lab
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions

July 12
Wet lab
· Digestion of FABP1 promoter PCR product
· Ran gel for PCR check of PPAR-alpha promoter
· Digest pEGFP-n1 for BBa_J52034 fadR
· Ran gel for pEGFP-n1 for BBa_J52034 fadR followed by gel purification
· BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and PfadBA
Dry Lab
Primers design for pCMV cloning for fadR expression

Week 3

July 15
Wet lab
· Ligation of FABP1 promoter, EGFP, and BBa_J176171, using 3 pieces ligation
· Transformation of FABP1 promoter, EGFP, and BBa_J176171 ligation
· FABP1 promoter+EGFP +BBa_J176171 ligation restriction check

July 16
Wet Lab
· Miniprep for full construct of FABP1 promoter and pEGFP-N1
· fadR and pEGFP-N1 Backbone parts ligation
· Plasmids extraction for pCMV cloning for fadR
· BBa_K817002 PfadBA promoter extraction by EcoR1 and Pst1 HF
· BBa_J52034 restriction by EcoR1 and Pst1 HF
· Inoculations for full construct of FABP1 promoter and pEGFP-N1
· Streak colonies containing the right construct for FABP1 promoter

July 17
Wet lab
· Plasmid extraction for FABP1 promoter+EGFP+BBa_J176171 by miniprep
· Repeat fadR and PfadBA constructs
· Digest pEGFP-n1 and BBa-J176171 for fadR
· Digestion for BBa_K817002 (PfadBA) extraction
· Gel check and gel extraction for BBa_J176171 for fadR and BBa_K817002 (PfadBA)

July 18
Wet lab
· Gel purification for all digested products
· FABP1 promoter digestion check for whole construct prior transfection
· PfadBA vector de phosphorylation and ligation with EGFP and BBa_J176171
· Transformation of PfadBA+EGFP+BBa_J176171

July 19
Wet lab
· FABP1 promoter preparation for transfection
· Ligation of PfadBA and BBa_J176171 followed by transformation
· PCR for PPAR-alpha promoter cloning
Dry lab
· Design for characterization of the transfected cells with FABP1 construct
· Review for Multiple Sites Mutagenesis
· Ordered primers for pCMV cloning from pEGFP-N1

Week 4

July 22
Wet lab
· FABP1 construct given for characterization and transfection
· Further colony screening for FABP1 construct, inoculations
· Inoculation of PfadBA and BBa_J176171
· Ran gel for PPAR-alpha promoter PCR products

July 23
Wet lab
· Primers arrival for pCMV cloning
· PCR for pCMV cloning
· Digestion and gel for construction check for FABP1 promoter
· PfadBA gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation
· Digestion of pEGFP-N1 for fadR, followed by gel check and extraction

July 24
Wet lab
· DNA purification from gel extraction for digested pEGFP-N1
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel for pCMV cloning from pEGFP-N1
· PCR clean up for pCMV cloning from pEGFP-N1
· PCR for PPAR-alpha promoter with new primers using Taq polymerase
· Transformation of mutagenesis products
· Inoculation of PfadBA ligation colonies

July 25
Wet lab
· Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase
· Minprep of PfadBA+EGFP+BBa_J176171 ligation colonies, followed by digestion check

July 26
Wet lab
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel before parental string digestion of mutagenesis products
· Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check
· Plasmids preparation according to transfection requirements, construct and controls

Week 5

July 30
Wet lab
· PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1
· BBa_J176171 vector de phosphorylation, followed by ligation with EGFP using T4 Ligase, then transformation into E. Coli strain DH10b
· PCR for PPAR-alpha promoter with new primers using Vent polymerase
Dry lab
· Discussion and re assessment of constructs related to pEGFP-N1

July 31
· Ran gel check for PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up
· PCR for PPAR-alpha promoter with reference primers using Vent polymerase
· Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase

August 2013

Week 1

August 2
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol

Week 2

August 5
Wet lab
· Gel Check for PPAR-alpha promoter PCR promoter cloning, using temperature gradient and every available set of primers designed.
· Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with fadR followed by transformation
· Ligation check of PfadBA+BBa_J176171+EGFP using Age1
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase
· gDNA Gel check
Dry lab
· Primers design for BBa_J04450-PSB1C3 Xba1 restriction site removal by site direct mutagenesis

August 6
Wet lab
· Ran gel for PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase
· Inoculation of fadR+BBa_J176171

August 7
Wet lab
· Digestion check of BBa_J176171+EGFP by Not1 and Pst1
· Digestion check of BBa_J176171+fadR by Xba1 and HindIII
· Ran gel for digestion check
· Inoculation of fadR+BBa_J176171, colony screening

August 8
Wet lab
· Miniprep for fadR+BBa_J176171, colony screening
· Digestion check of BBa_J176171+fadR by Xba1 and HindIII
· Ran gel for digestion check
· Inoculation of fadR+BBa_J176171, colony screening
· pCMV from cloned from pEGFP-N1adding restriction sites for ligation with EGFP and BBa_J176171, followed by transformation

August 9
Wet lab
· Primers arrival for BBa_J04450-PSB1C3 for site direct mutagenesis
· Digestion check of BBa_J176171+fadR Xba1 and HindIII, Xba1 and Spe1
· Ran gel for digestion check
· Site direct Mutagenesis for BBa_J04450-PSB1C3 removing Xba1 restriction site, followed by gel check before parental string digestion and transformation
· Inoculation of pCMV from cloned from pEGFP-N1adding restriction sites for ligation with EGFP and BBa_J176171

Week 3

 

August 12
Wet lab
·      Miniprep of pCMV+EGFP+ BBa_J176171, followed by digestion check and gel
·      PCR for pCMV cloning from pEGFP-N1 using Vent polymerase, followed by gel check and PCR clean up
·      Digestion check of BBa_J176171+fadR by Xba1, HindIII and Spe1
·      Ran gel for digestion check
·      pEGFP-N1 digestion for GRP78, extraction pCMV using Ase1 and Xho1, followed by gel check and DNA purification
·      Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with fadR followed by transformation
·      Site direct mutagenesis for BBa_J04450-PSB1C3, followed by gel check before parental digestion with Dpn1 and transformation

 

August 13
Wet lab
·      Primers phosphorylation for FABP1 promoter Multi-site Mutagenesis using T4 PNK
·      Primers phosphorylation for Site Direct Mutagenesis for BBa_J04450-PSB1C3 using T4 PNK
·      Site Direct Mutagenesis for BBa_J04450-PSB1C3 using phosphorylated primers and non phosphorylated primers
·      Overnight culture for Competent cells
·      pDRIVE_hGRP78 arrival, transformation into E. Coli strain DH10b  

 

August 14
Wet lab
·      Nothing done due the typhoon

 

August 15
Wet lab
·      PfadBA+pEGFP-N1 ligation, first vector dephosphorylation using Antarctic phosphatase followed by ligation by T4Ligase and transformation into E. Coli SURE strain.
·      pCMV+EGFP+BBa_J176171 digestion check, followed by gel check
·      Repeat GRP78+pEGFP-N1 ligation, followed by transformation into E. Coli DH10b strain.
·      Digestion check for fadR+BBa_J176171 usign Xba1 and HindIII
·      Inoculations of fadR+ BBa_J176171, pCMV+EGFP+ BBa_J176171, PfadBA+pEGFP-N1, PfadBA+EGFP+ BBa_J176171 followed by gel check

 

August 16
Wet lab
·      Miniprep of fadR+ BBa_J176171, pCMV+EGFP+ BBa_J176171, PfadBA+pEGFP-N1, PfadBA+EGFP+ BBa_J176171
·      Restriction check for fadR+ BBa_J176171 and PfadBA+pEGFP-N1 followed by gel check
·      Inoculations of fadR+ BBa_J176171, PfadBA+pEGFP-N1, PfadBA+EGFP+ BBa_J176171
·      DH105alpha E. Coli competent cells preparation

 

Week 4

 

August 19
Wet lab
·      Repeat GRP78+pEGFP-N1 ligation, followed by transformation into E. Coli DH10b strain.
·      Miniprep of fadR+ BBa_J176171, PfadBA+pEGFP-N1, PfadBA+EGFP+ BBa_J176171
·      Restriction check for PfadBA+pEGFP-N1 followed by gel check
·      Restriction check for fadR+ BBa_J176171 and PfadBA+pEGFP-N1 followed by gel check
·      Streak colony with the right fadR+BBa_J176171 construct
·      DH5alpha E. Coli competent cells preparation
·       

August 20
Wet lab
·      Digestion check PfadBA+pEGFP-N1 with Pst1, Xba1. Followed by gel check
·      Digestion check for damaged plates of GRP78+pEGFP-N1 using Xba1
·      Digestion check of PfadBA+EGFP+ BBa_J176171 using Age1HF, HindIII and Xba1
·      Site Direct Mutagenesis for illegal restriction sites for FABP1 promoter
·      Inoculate GRP78+pEGFP-N1, pCMV+EGFP+BBa_J176171
·      DH10b E. Coli competent cells preparation

 

August 21
Wet lab
·      Miniprep GRP78+pEGFP-N1, followed by digestion check and gel check using Spe1
·      Miniprep pCMV+EGFP+BBa_J176171 followed by digestion check using Nde1 and Age1HF
·      Digestion check of PfadBA+EGFP+BBa_J176171 using Nde1 , fadR+ BBa_J176171 using Age1
·      Site direct Mutagenesis for FABP1 promoter+EGFP+BBa_J176171
·      PCR for GRP78 promoter cloning from pDRIVE_hGRP78, followed by gel check PCR clean up, digestion using Ase1 and Xho1 and DNA purification

 

August 22
Wet lab
·      DH10b E. Coli competent cells preparation
·      Site direct Mutagenesis for FABP1 promoter+EGFP+BBa_J176171 with phosphorylated primers, followed by gel check before Dpn1 parental string digestion
·      Ligation of GRP78 and pEGFP-N1, first vector dephophorylation and ligation with T4Ligase. Transformed into E. Coli DH10b strain
·      Transformation of pBlueScript KS (+) in to SURE E. Coli
Dry lab
·      Discuss about FABP1 construct vanishing under PCR conditions

 

 

Week 5

 

August 26
Wet lab
·      FABP1 promoter assessment under PCR conditions
·      BBa_J176171+EGFP assessment under PCR conditions
·      BBa_J176171 assessment under PCR conditions
·      Temperature gradient for FABP1 promoter+BBa_J176171+EGFP construct
·      Inoculate pBlueScript KS (+)

 

August 27
Wet lab
·      DH10b E. Coli competent cells preparation

 

August 28
Wet lab
·      BBa_J176171 assessment under PCR conditions
·      Temperature gradient for FABP1 promoter+BBa_J176171+EGFP construct
·      PCR for FABP1 promoter cloning from gDNA
·      GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into E.Coli DH10b
·      Miniprep pBlueScript KS (+)
·      Digest pBlueScript KS (+) with Xba1 and BamH1HF
·      Restreak BBa_J176171 and inoculate it

 

August 29
Wet lab
·      Miniprep estreaked BBa_J176171
·      GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into E.Coli DH10b
·      Ligation of FABP1 promoter in to pBlueScript KS (+) using T4 Ligase followed by transformation into DH10b E. Coli
·      Transformation of BBa_J176171 into SURE E. Coli

 

August 30
Wet lab
·      Inoculate GRP78+pEGFP-N1
·      Inoculate FABP1 promoter+pBlueScript KS (+)
·      Inoculate BBa_J176171
·      PCR conditions check for BBa_J176171 looking for heat degradation

 

August 31
Wet lab
·      Miniprep of FABP1 promoter+pBlueScript KS (+), BBa_J176171
·      PCR conditions check for BBa_J176171 looking for heat degradation


September 2013

Week 1

 

September 1
Wet lab
·      Digestion check for FABP1 promoter+pBlueScript KS (+) using EcoR1. Followed by gel check.

 

September 2
Wet lab
·      Digestion check for FABP1 promoter+pBlueScript KS (+) using EcoR1. Followed by gel check.

 

September 3
Wet lab
·      Restriction check from the damaged plates GRP78+pEGFP-N1 using Xba1
·      Repeat FABP1 promoter and pBlueScript KS (+) ligation, vector Dephosphorylation by Antartic Phosphatase and ligation using T4 Ligase, followed by transformation into DH10b E. Coli

 

September 4
Wet lab
·      GRP78 promoter PCR Cloning from pDRIVE_hGFRP78, followed by PCR cleanup using kit. Then restriction check using Xba1
·      pEGFP-N1 restriction for GRP78 assembly, digested by Ase1 and BamH1, then Gel check, Gel extraction and purification, vector dephosphorilation and ligation by T4 ligase. Transformed into DH10b E. Coli
·      Inoculation of FABP1 promoter+ pBlueScript KS (+)

 

September 5
Wet lab
·      Miniprep of FABP1 promoter+pBlueScript KS (+) then restriction check using Ecor1, followed by gel check.
·      Site Direct Mutagenesis for FABP1 promoter+pBlueScript KS (+), Gel Check

 

September 6
Wet lab
·      Restriction of GRP78 PCR product by Ase1 and Xho1
·      PCR for PPAR-alpha promoter cloning
Dry lab
·      Review on previous Mutagenesis attempts and troubleshooting

 

Week 2

 

September 9
Wet lab
·      Ran gel for PPAR-alpha promoter cloning
·      pEGFP-N1  digestion for PPAR-alpha promoter and vector dephosphorylation

 

September 10
Wet lab
·      Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check
·      Site Direct Mutagenesis FABP1 promoter+pBlueScript KS(+) then Gel Check

 

September 11
Wet lab
·      Site Direct Mutagenesis FABP1 promoter+pBlueScript KS(+) then Gel Check
·      Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check
·      Digest mutant FABP1 promoter+ pBlueScript KS(+) with Dpn1 then transformed into SURE E. Coli

 

September 12
Wet lab
·      GRP78 PCR product digested by Ase1 and Xho1, ligated with dephosphorylated pEGFP-N1 vector and ligated by T4 ligase, transformed into DH10b E. Coli
·      Inoculations of mutant FABP1 promoter+pBlueScript KS(+)

 

September 13
Wet lab
·      Miniprep of Mutant FABP1 promoter+ pBlueScript KS(+), then restriction check using Ecor1, ran gel
·       Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check

 

September 14
Wet lab
·       Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check

 

Week 3
Content 1
Week 4
Content 1
Week 5
Content 1