Team:Hong Kong HKUST/notebook/mod1

From 2013.igem.org

(Difference between revisions)
 
(22 intermediate revisions not shown)
Line 3: Line 3:
   <style>
   <style>
     * { margin:0; padding:0; } /* a simple reset */
     * { margin:0; padding:0; } /* a simple reset */
-
     .head, li, h2 { margin-bottom:15px;z-index:-11;font-family:"Trebuchet MS", Helvetica, sans-serif }
+
     .head, li, h2 { margin-bottom:15px;z-index:-11; }
     .head { display:block; background:#35BB91;border-radius:500px;margin-bottom:5px;align:center;width:90%;z-index:-3;font-family:"Trebuchet MS", Helvetica, sans-serif }
     .head { display:block; background:#35BB91;border-radius:500px;margin-bottom:5px;align:center;width:90%;z-index:-3;font-family:"Trebuchet MS", Helvetica, sans-serif }
     .content { display:none; background:#8FF5D5;border-radius:100px;margin-bottom:5px;align:center;width:90%;z-index:-3;padding-left:100px;padding-right:100px;font-family:"Trebuchet MS", Helvetica, sans-serif}
     .content { display:none; background:#8FF5D5;border-radius:100px;margin-bottom:5px;align:center;width:90%;z-index:-3;padding-left:100px;padding-right:100px;font-family:"Trebuchet MS", Helvetica, sans-serif}
     li { position:relative;overflow:hidden; }
     li { position:relative;overflow:hidden; }
 +
#iGEM_Logo{
 +
width:100px;
 +
height:80px;
 +
position:absolute;
 +
right:10px;
 +
top:60px;
 +
z-index:+15;
 +
}
 +
#hkust_Logo{
 +
width:60px;
 +
height:80px;
 +
position:absolute;
 +
right:110px;
 +
top:60px;
 +
z-index:+15;
 +
}
   </style>
   </style>
   <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>
Line 46: Line 62:
});
});
</script>
</script>
 +
<style>
 +
.pos_fixed
 +
{
 +
position:fixed;
 +
top:150px;
 +
left:10px;
 +
}
 +
 +
ol.pos_fixed
 +
{
 +
list-style-type:none;
 +
margin:0;
 +
padding:0;
 +
}
 +
.pos_fixed a:link,.pos_fixed a:visited
 +
{
 +
display:block;
 +
font-weight:bold;
 +
color:#FFFFFF;
 +
background-color:#E32E51;
 +
width:120px;
 +
text-align:center;
 +
padding:4px;
 +
text-decoration:none;
 +
text-transform:uppercase;
 +
}
 +
.pos_fixed a:hover,.pos_fixed a:active
 +
{
 +
background-color:#E32E51;
 +
}
 +
</style>
<style type="text/css">
<style type="text/css">
-
body{background-color:#d5cfbf;width:100%;margin-bottom:20px;min-width:600px;max-width:2000px;height:100%;font: x-small sans-serif;}
+
body{background-color:#494042;width:100%;margin-bottom:20px;min-width:600px;max-width:2000px;height:100%;font: x-small sans-serif;}
#menubar  
#menubar  
{background-color:#EEECEC;opacity:0.8;top:-4px;width:360px;height:23px;position:absolute;top:-20px;}
{background-color:#EEECEC;opacity:0.8;top:-4px;width:360px;height:23px;position:absolute;top:-20px;}
Line 238: Line 285:
}
}
#flight{z-index:-2;}
#flight{z-index:-2;}
 +
h3{font-size:28px;font-family:"Trebuchet MS", Helvetica, sans-serif;}
 +
#title{border: solid 2px;border-radius:15px;width:90%;padding:15px;margin-left:50px;margin-bottom:25px;}
</style>
</style>
</head>
</head>
<body>
<body>
 +
<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>
 +
         
 +
 +
<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>
 +
<ol class="pos_fixed">
 +
<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>
 +
</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>
Line 254: Line 314:
<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>
+
<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>
+
<li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/modules">Modules Description</a></li>
-
<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/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>
+
<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>
+
<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>
Line 270: Line 333:
<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>
+
<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>
Line 285: Line 349:
<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>
+
<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>
+
<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>
+
<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>
+
<br><br><br><br><br><br><br><br>
 +
<div id="title"><center><h3>FA Quantification and Cell Viability 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>
Line 376: Line 444:
<ul>
<ul>
   <li>Repeated GCMS: No accurate results</li>
   <li>Repeated GCMS: No accurate results</li>
-
   <li>Changed Medium for the cell line</li>
+
   <li>Changed medium for the cell line</li>
   <li>Checked lipofectamine protocol</li>
   <li>Checked lipofectamine protocol</li>
</ul>
</ul>
Line 382: Line 450:
<p><strong>July 11:</strong></p>
<p><strong>July 11:</strong></p>
<ul>
<ul>
-
   <li>GCMS again for the same sodium palmitate we made: Not accurate results</li>
+
   <li>GCMS again for the same sodium palmitate we made: No accurate results</li>
   <li>Passaged HepG2 cell line</li>
   <li>Passaged HepG2 cell line</li>
</ul>
</ul>
Line 421: Line 489:
<p><strong>July 18:</strong></p>
<p><strong>July 18:</strong></p>
<ul>
<ul>
-
   <li>Seeded HepG2 cells in 24-well plate for transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP</li>
+
   <li>Seeded HepG2 cells in 24-well plate for transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li>
   <li>Got polylysine for coating</li>
   <li>Got polylysine for coating</li>
</ul>
</ul>
Line 443: Line 511:
<p><strong>July 22:</strong></p>
<p><strong>July 22:</strong></p>
<ul>
<ul>
-
   <li>Transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP on 96-well plate</li>
+
   <li>Transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP on 96-well plate</li>
   <li>Added gradient concentration of sodium palmitate into HepG2 cells for MTT assay</li>
   <li>Added gradient concentration of sodium palmitate into HepG2 cells for MTT assay</li>
   <li>Coated coverslips with polylysine for HepG2 cell staining and fixation</li>
   <li>Coated coverslips with polylysine for HepG2 cell staining and fixation</li>
Line 450: Line 518:
<p><strong>July 23:</strong></p>
<p><strong>July 23:</strong></p>
<ul>
<ul>
-
   <li>Replaced the medium after transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP</li>
+
   <li>Replaced the medium after transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li>
   <li>Finished MTT assay to get cell viability in different FA concentrations in 24 hours</li>
   <li>Finished MTT assay to get cell viability in different FA concentrations in 24 hours</li>
-
   <li>Transferredcells containing PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP into 24-well plate with well-coated coverslips in each well</li>
+
   <li>Transferred cells containing PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into 24-well plate with well-coated coverslips in each well</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>July 24:</strong></p>
<p><strong>July 24:</strong></p>
<ul>
<ul>
-
   <li>Stained mitochondria by Mito-tracker@Red and fixating by formaldehyde of HepG2 cells transfected with PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP in 24-well plate</li>
+
   <li>Stained mitochondria by Mito-tracker@Red and fixating by formaldehyde of HepG2 cells transfected with pEGFP-N1, pCMV/myc/mito/EGFP and <i>Pmv</i>/myc/mito/GFP in 24-well plate</li>
-
   <li>Passaged HepG2 cells and changing medium of the cell line</li>
+
   <li>Passaged HepG2 cells and changed medium of the cell line</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 469: Line 537:
<ul>
<ul>
   <li>Coated coverslips by polylysine</li>
   <li>Coated coverslips by polylysine</li>
-
   <li>Seeded HepG2 cells in one 24-well plate for transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP </li>
+
   <li>Seeded HepG2 cells in one 24-well plate for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP </li>
-
   <li>Seeded HepG2 cells on two 96-well plate, for transfection of FABP and MTT assay in 48 hours</li>
+
   <li>Seeded HepG2 cells on two 96-well plate, for transfection of FABP construct and MTT assay in 48 hours</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 482: Line 550:
<p><strong>July 29:</strong></p>
<p><strong>July 29:</strong></p>
<ul>
<ul>
-
   <li>Transfection of FABP with PEGFP-N1 as control</li>
+
   <li>Transfection of FABP construct with pEGFP-N1 as control</li>
-
   <li>Transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP in 24-well plate with well-coated coverslips</li>
+
   <li>Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24-well plate with well-coated coverslips</li>
   <li>Added gradient concentration form 0.1mM to 1.2mM of FA into the plate for MTT assay</li>
   <li>Added gradient concentration form 0.1mM to 1.2mM of FA into the plate for MTT assay</li>
</ul>
</ul>
Line 489: Line 557:
<p><strong>July 30:</strong></p>
<p><strong>July 30:</strong></p>
<ul>
<ul>
-
   <li>Changed medium after transfection of PEGFP-N1 and FABP and seeing the results: Both PEGFP-N1 and FABP did not show GFP signals</li>
+
   <li>Changed medium after transfection of pEGFP-N1 and FABP construct and saw the results: Both pEGFP-N1 and FABP construct did not show GFP signals</li>
-
   <li>Changed medium after transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP</li>
+
   <li>Changed medium after transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li>
-
   <li>Did staining and fixation of the coverslips and seeing the results: No obvious overlapping patterns of GFP signals and Mito-tracker red</li>
+
   <li>Did staining and fixation of the coverslips and saw the results: No obvious overlapping patterns of GFP signals and Mito-tracker red</li>
   <li>Passaged HepG2 cells</li>
   <li>Passaged HepG2 cells</li>
</ul>
</ul>
Line 507: Line 575:
<p><strong>Aug 1:</strong></p>
<p><strong>Aug 1:</strong></p>
<ul>
<ul>
-
   <li>Seeded cells for transfection of PEGFP-N1, FABP, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP</li>
+
   <li>Seeded cells for transfection of pEGFP-N1, FABP construct, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li>
   <li>Changed medium for HepG2 cell line</li>
   <li>Changed medium for HepG2 cell line</li>
   <li>Got results for MTT assay in 48 hours</li>
   <li>Got results for MTT assay in 48 hours</li>
Line 515: Line 583:
   <li>Checked if transfection (after 22 hrs) worked </li>
   <li>Checked if transfection (after 22 hrs) worked </li>
   <ul>
   <ul>
-
     <li>No GFP signal for PEFGP-N1, PCMV/myc/mito/EGFP and PCMV/myc/mito/GFP</li>
+
     <li>No GFP signal for pEFGP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li>
   </ul>
   </ul>
   <p>&nbsp;</p>
   <p>&nbsp;</p>
Line 540: Line 608:
<ul>
<ul>
   <li>Observe transfected cell. GFP signal expressed </li>
   <li>Observe transfected cell. GFP signal expressed </li>
-
   <li>Seeded cells for transfection of PEGFP-N1, and (FABP/EGFP) in 96 well-plate </li>
+
   <li>Seeded cells for transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well-plate </li>
-
   <li>Seeded cells for transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMV/myc/mito/GFP in 24 well plate with polylysine coated coverslips </li>
+
   <li>Seeded cells for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24 well plate with polylysine coated coverslips </li>
   <li>Seeded cells for MTT assay </li>
   <li>Seeded cells for MTT assay </li>
</ul>
</ul>
Line 547: Line 615:
<p><strong>Aug 8:</strong></p>
<p><strong>Aug 8:</strong></p>
<ul>
<ul>
-
   <li>Observed transfection of PEGFP-N1, and (FABP/EGFP) in 96 well-plate. No GFP signal observed</li>
+
   <li>Observed transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well-plate. No GFP signal observed</li>
-
   <li>Observed transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMV/myc/mito/GFP. GFP signal observed</li>
+
   <li>Observed transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP. GFP signal observed</li>
-
   <li>Stained mitochondria of cells transfected with PEGFP-N1, PCMV/myc/mito/EGFP and PCMV/myc/mito/GFP using mito tracking dye </li>
+
   <li>Stained mitochondria of cells transfected with pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP using mito tracking dye </li>
   <li>Observed cell with both transfection and stained mitochondria: No GFP signal observed because transfection occurred with very low efficiency. </li>
   <li>Observed cell with both transfection and stained mitochondria: No GFP signal observed because transfection occurred with very low efficiency. </li>
-
   <li>Prepared new cell line: HEK 293 FT </li>
+
   <li>Prepared new cell line: HEK293FT </li>
   <li>Conducted MTT assay </li>
   <li>Conducted MTT assay </li>
</ul>
</ul>
Line 563: Line 631:
<p><strong>Aug 12: </strong></p>
<p><strong>Aug 12: </strong></p>
<ul>
<ul>
-
   <li>MTT Assay without seeding ad incubation </li>
+
   <li>MTT Assay without seeding and incubation </li>
-
   <li>Changed medium for HepG2 and HEK 293 FT cell lines </li>
+
   <li>Changed medium for HepG2 and HEK293FT cell lines </li>
   <li>Prepared polylysine coated coverslips </li>
   <li>Prepared polylysine coated coverslips </li>
</ul>
</ul>
Line 570: Line 638:
<p><strong>Aug 13:</strong></p>
<p><strong>Aug 13:</strong></p>
<ul>
<ul>
-
   <li>Seeded cells using HEK 293 FT cell line for transfection </li>
+
   <li>Seeded cells using HEK293FT cell line for transfection </li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 580: Line 648:
<p><strong>Aug 16:</strong></p>
<p><strong>Aug 16:</strong></p>
<ul>
<ul>
-
   <li>Seeded cells HEK 293 FT cell line for transfection </li>
+
   <li>Seeded cells HEK293FT cell line for transfection </li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 591: Line 659:
<p><strong>Aug 19:</strong></p>
<p><strong>Aug 19:</strong></p>
<ul>
<ul>
-
   <li>Transfection of PEGFP-N1, and (FABP/EGFP) in 96 well plate and PEGFP-N1, PCMV/myc/mito/EGFP and PCMV/myc/mito/GFP in 24 well plate.</li>
+
   <li>Transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well plate and pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24 well plate.</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Aug 20:</strong></p>
<p><strong>Aug 20:</strong></p>
<ul>
<ul>
-
   <li>Seeing results of transfection of FABP and PEGFP-N1 in HEK 293FT cells and seeing the results: PEGFP-N1 had signal but FABP did not show GFP signals</li>
+
   <li>Seeing results of transfection of FABP construct and pEGFP-N1 in HEK293FT cells and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Aug 22:</strong></p>
<p><strong>Aug 22:</strong></p>
<ul>
<ul>
-
   <li>Transfection of PEGFP-N1 and FABP into HEK 293FT cells</li>
+
   <li>Transfection of pEGFP-N1 and FABP construct into HEK293FT cells</li>
-
   <li>Changing medium for HEK 293FT cells  and HepG2 cells and seeing the results: PEGFP-N1 had signal but FABP did not show GFP signals</li>
+
   <li>Changing medium for HEK293FT cells and HepG2 cells and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 614: Line 682:
<p><strong>Aug 26:</strong></p>
<p><strong>Aug 26:</strong></p>
<ul>
<ul>
-
   <li>Transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP in 60mm TC plates</li>
+
   <li>Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 60mm TC plates</li>
-
   <li>Changing medium of transfection of the previous day and seeing the results: PEGFP-N1 had signal but FABP did not show GFP signals</li>
+
   <li>Changing medium of transfection of the previous day and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 627: Line 695:
<p><strong>Aug 29:</strong></p>
<p><strong>Aug 29:</strong></p>
<ul>
<ul>
-
   <li>Transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP into HEK293FT cells</li>
+
   <li>Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into HEK293FT cells</li>
-
   <li>Maintaining HEK 293FT and HepG2 cell line</li>
+
   <li>Maintaining HEK293FT and HepG2 cell line</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Aug 30:</strong></p>
<p><strong>Aug 30:</strong></p>
<ul>
<ul>
-
   <li>Staining and fixation for HEK cells transfected by PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP</li>
+
   <li>Staining and fixation for HEK cells transfected by pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li>
-
   <li>Seeing the results for transfected HEK cells: No obvious overlapping pattern of GFP signals and Mito-tracker</li>
+
   <li>Seeing the results for transfected HEK293FT cells: No obvious overlapping pattern of GFP signals and Mito-tracker</li>
-
   <li>Seeding HepG2 cells for Transfection of FABP and PEGFP-N1 in 96-well plate</li>
+
   <li>Seeding HepG2 cells for Transfection of FABP construct and pEGFP-N1 in 96-well plate</li>
-
   <li>Seeding HEK 293FT cells for transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP</li>
+
   <li>Seeding HEK 293FT cells for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li>
-
   <li>Passaging HEK cell line</li>
+
   <li>Passaging HEK293FT cell line</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Aug 31:</strong></p>
<p><strong>Aug 31:</strong></p>
<ul>
<ul>
-
   <li>Transfection with PEGFP-N1 and PCMV/PolyA/EGFP into HEK 293FT cells</li>
+
   <li>Transfection with pEGFP-N1 and pCMV/PolyA/EGFP into HEK293FT cells</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 656: Line 724:
<p><strong>Sept 1:</strong></p>
<p><strong>Sept 1:</strong></p>
<ul>
<ul>
-
   <li>Checking results for transfection on Aug 31: PEGFP had strong signal while PolyA did not have signals</li>
+
   <li>Checking results for transfection on Aug 31: pEGFP-N1 had strong signal while PolyA did not have signals</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Sept 2:</strong></p>
<p><strong>Sept 2:</strong></p>
<ul>
<ul>
-
   <li>Transfection of FABP and PEGFP-N1 in HepG2 cells</li>
+
   <li>Transfection of FABP construct and pEGFP-N1 in HepG2 cells</li>
-
   <li>Check results for HEK 293FT cells been transfected by PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP, by using confocal microscope</li>
+
   <li>Check results for HEK293FT cells been transfected by pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP, by using confocal microscope</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Sept 3:</strong></p>
<p><strong>Sept 3:</strong></p>
<ul>
<ul>
-
   <li>Adding FA into HepG2 cells been transfected by FABP and PEGFP-N1</li>
+
   <li>Adding FA into HepG2 cells been transfected by FABP construct and pEGFP-N1</li>
-
   <li>Maintaining HepG2 and HEK cell line</li>
+
   <li>Maintaining HepG2 and HEK293FT cell line</li>
-
   <li>Drug selection Test on HEK 293FT test by purmycin</li>
+
   <li>Drug selection Test on HEK293FT test by purmycin</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Sept 4:</strong></p>
<p><strong>Sept 4:</strong></p>
<ul>
<ul>
-
   <li>Drug selection test on HEK 293 cells by neomycin and puromycin</li>
+
   <li>Drug selection test on HEK293FT cells by neomycin and puromycin</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Sept 5:</strong></p>
<p><strong>Sept 5:</strong></p>
<ul>
<ul>
-
   <li>Staining and fixation of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP and seeing he results by confocal microscope</li>
+
   <li>Staining and fixation of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP and seeing he results by confocal microscope</li>
   <li>Making mew DMEM</li>
   <li>Making mew DMEM</li>
   <li>Drug selection test observation</li>
   <li>Drug selection test observation</li>
Line 686: Line 754:
<p><strong>Sept 6:</strong></p>
<p><strong>Sept 6:</strong></p>
<ul>
<ul>
-
   <li>Transfection of FAPB and PEGFP-N1 into HepG2 cells in 60 mm plates</li>
+
   <li>Transfection of FAPB and pEGFP-N1 into HepG2 cells in 60 mm plates</li>
-
   <li>Maintaining cell line for HEK cells</li>
+
   <li>Maintaining cell line for HEK293FT cells</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Sept 7:</strong></p>
<p><strong>Sept 7:</strong></p>
<ul>
<ul>
-
   <li>Checking results for transfection on Sept 6: PEGFP-N1 showed weak GFP signal, no signals for FABP</li>
+
   <li>Checking results for transfection on Sept 6: pEGFP-N1 showed weak GFP signal, no signals for FABP</li>
   <li>Change medium for drug selection test</li>
   <li>Change medium for drug selection test</li>
</ul>
</ul>
Line 698: Line 766:
<p><strong>Sept 8:</strong></p>
<p><strong>Sept 8:</strong></p>
<ul>
<ul>
-
   <li>Maintaining cell line for HEK 293FT cells and HepG2 cells</li>
+
   <li>Maintaining cell line for HEK293FT cells and HepG2 cells</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
Line 709: Line 777:
<p><strong>Sept 9:</strong></p>
<p><strong>Sept 9:</strong></p>
<ul>
<ul>
-
   <li>Checking transfection for FABP again: Weak signals were shown from the debris of the cells</li>
+
   <li>Checking transfection for FABP construct again: Weak signals were shown from the debris of the cells</li>
-
   <li>Transfection of AceA</li>
+
   <li>Transfection of <i>AceA</i> construct</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Sept 11:</strong></p>
<p><strong>Sept 11:</strong></p>
<ul>
<ul>
-
   <li>Seeding HEK cells into 35mm plates for transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP</li>
+
   <li>Seeding HEK293FT cells into 35mm plates for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li>
-
   <li>Cell splitting of AceA</li>
+
   <li>Cell splitting of ACE</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>
<p><strong>Sept 12:</strong></p>
<p><strong>Sept 12:</strong></p>
<ul>
<ul>
-
   <li>Transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP into HEK 293 FT cells</li>
+
   <li>Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into HEK293FT cells</li>
</ul>
</ul>
<p>&nbsp;</p>
<p>&nbsp;</p>

Latest revision as of 23:13, 27 September 2013

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








FA Quantification and Cell Viability Module's Notebook

June 2013

Week 4

 

June 25:

  • Prepared DMEM in TC room
  • Added FBS (Serum) & Antibiotics into DMEM

 

June 27:

  • Designed the experiment for testing FA concentration by GCMS

 

June 28:

  • HepG2 cell culturing (first generation)

July 2013

Week 1

 

July 2:

  • HepG2 cell passaging
  • Made sodium palmitate from powder to solution by 50% Ethanol

 

July 3:

  • Used GCMS to quantify the FA in the medium

 

July 4:

  • Passaged HepG2 cells
  • Changed medium for cell line
  • Searched for information of MTT assay and Transfection by lipofectamine
  • Got GCMS results for the last day: Not accurate/usable

 

July 5:

  • Repeated GCMS test again testing FA in medium and we made
  • Got the result of GCMS: Not accurate/usable

 

Week 2

 

July 8:

  • Passaged HepG2 cells
  • Used some HepG2 cells to extract genomic DNA

 

July 9:

  • Another try of GCMS with a gradient concentration of FA
  • Got the result of GCMS: Not match with the concentration we made

 

July 10:

  • Repeated GCMS: No accurate results
  • Changed medium for the cell line
  • Checked lipofectamine protocol

 

July 11:

  • GCMS again for the same sodium palmitate we made: No accurate results
  • Passaged HepG2 cell line

 

July 12:

  • Prepared reagents for MTT assay for cell viability test, protocols checked
  • Changed the medium of the cell line

 

Week 3

 

July 15:

  • Seeded HepG2 cells into 96-well plate for MTT cell viability test and adding FA
  • Seeded HepG2 cells into 96-well plate for transfection of PEGFP-N1

 

July 16:

  • Transfection of PEGFP-N1 into HepG2 cells
  • Passaged HepG2 cells

 

July 17:

  • Changed the medium after transfection had been done in 24 hours
  • Did MTT assay and getting result (adding FA in the wrong time, cannot be used)
  • Made new DMEM
  • Changed medium for the cell line

 

July 18:

  • Seeded HepG2 cells in 24-well plate for transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
  • Got polylysine for coating

 

July 19:

  • Changed the medium for the HepG2 cell line
  • Seeded cells for another MTT assay
  • Checked HepG2 cells for transfection: growing slow, 30% confluency, cannot do transfection
  • Seeded cells on a 96-well plate for transfection in the coming week
  • Passaged HepG2 cells

 

Week 4

 

July 22:

  • Transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP on 96-well plate
  • Added gradient concentration of sodium palmitate into HepG2 cells for MTT assay
  • Coated coverslips with polylysine for HepG2 cell staining and fixation

 

July 23:

  • Replaced the medium after transfection of PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
  • Finished MTT assay to get cell viability in different FA concentrations in 24 hours
  • Transferred cells containing PEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into 24-well plate with well-coated coverslips in each well

 

July 24:

  • Stained mitochondria by Mito-tracker@Red and fixating by formaldehyde of HepG2 cells transfected with pEGFP-N1, pCMV/myc/mito/EGFP and Pmv/myc/mito/GFP in 24-well plate
  • Passaged HepG2 cells and changed medium of the cell line

 

July 25:

  • Got results for cell staining and fixation: No obvious overlapping pattern between GPF signal and Mito-tracker red.

 

July 26:

  • Coated coverslips by polylysine
  • Seeded HepG2 cells in one 24-well plate for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
  • Seeded HepG2 cells on two 96-well plate, for transfection of FABP construct and MTT assay in 48 hours

 

Week 5

 

July 29:

  • Transfection of FABP construct with pEGFP-N1 as control
  • Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24-well plate with well-coated coverslips
  • Added gradient concentration form 0.1mM to 1.2mM of FA into the plate for MTT assay

 

July 30:

  • Changed medium after transfection of pEGFP-N1 and FABP construct and saw the results: Both pEGFP-N1 and FABP construct did not show GFP signals
  • Changed medium after transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
  • Did staining and fixation of the coverslips and saw the results: No obvious overlapping patterns of GFP signals and Mito-tracker red
  • Passaged HepG2 cells

 

August 2013

Week 1

 

Aug 1:

  • Seeded cells for transfection of pEGFP-N1, FABP construct, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
  • Changed medium for HepG2 cell line
  • Got results for MTT assay in 48 hours

Aug 2:

  • Checked if transfection (after 22 hrs) worked
    • No GFP signal for pEFGP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP

     

Week 2

 

Aug 5:

  • Transfection of HepG2 cells on 10cm Plates
  • Passaged P(n+8) HepG2 cells into one P(n+9)
  • Seeded HepG2 cells on 96-well plates for transfection

 

Aug 6:

  • Trouble shooting for transfection of HepG2 cells on 96 well plates

 

Aug 7:

  • Observe transfected cell. GFP signal expressed
  • Seeded cells for transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well-plate
  • Seeded cells for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24 well plate with polylysine coated coverslips
  • Seeded cells for MTT assay

 

Aug 8:

  • Observed transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well-plate. No GFP signal observed
  • Observed transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP. GFP signal observed
  • Stained mitochondria of cells transfected with pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP using mito tracking dye
  • Observed cell with both transfection and stained mitochondria: No GFP signal observed because transfection occurred with very low efficiency.
  • Prepared new cell line: HEK293FT
  • Conducted MTT assay

 

Week 3

 

Aug 12:

  • MTT Assay without seeding and incubation
  • Changed medium for HepG2 and HEK293FT cell lines
  • Prepared polylysine coated coverslips

 

Aug 13:

  • Seeded cells using HEK293FT cell line for transfection

 

Aug 15:

  • Cells detached and not in good condition for transfection

 

Aug 16:

  • Seeded cells HEK293FT cell line for transfection

 

Week 4

 

Aug 19:

  • Transfection of pEGFP-N1, and (FABP construct/EGFP) in 96 well plate and pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 24 well plate.

 

Aug 20:

  • Seeing results of transfection of FABP construct and pEGFP-N1 in HEK293FT cells and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals

 

Aug 22:

  • Transfection of pEGFP-N1 and FABP construct into HEK293FT cells
  • Changing medium for HEK293FT cells and HepG2 cells and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals

 

Week 5

 

Aug 26:

  • Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP in 60mm TC plates
  • Changing medium of transfection of the previous day and seeing the results: pEGFP-N1 had signal but FABP did not show GFP signals

 

Aug 28:

  • Changing medium of transfection of the previous day
  • Staining and fixation of
  • Drawing MTT standard curves

 

Aug 29:

  • Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into HEK293FT cells
  • Maintaining HEK293FT and HepG2 cell line

 

Aug 30:

  • Staining and fixation for HEK cells transfected by pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
  • Seeing the results for transfected HEK293FT cells: No obvious overlapping pattern of GFP signals and Mito-tracker
  • Seeding HepG2 cells for Transfection of FABP construct and pEGFP-N1 in 96-well plate
  • Seeding HEK 293FT cells for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
  • Passaging HEK293FT cell line

 

Aug 31:

  • Transfection with pEGFP-N1 and pCMV/PolyA/EGFP into HEK293FT cells

 

September 2013

Week 1

 

Sept 1:

  • Checking results for transfection on Aug 31: pEGFP-N1 had strong signal while PolyA did not have signals

 

Sept 2:

  • Transfection of FABP construct and pEGFP-N1 in HepG2 cells
  • Check results for HEK293FT cells been transfected by pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP, by using confocal microscope

 

Sept 3:

  • Adding FA into HepG2 cells been transfected by FABP construct and pEGFP-N1
  • Maintaining HepG2 and HEK293FT cell line
  • Drug selection Test on HEK293FT test by purmycin

 

Sept 4:

  • Drug selection test on HEK293FT cells by neomycin and puromycin

 

Sept 5:

  • Staining and fixation of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP and seeing he results by confocal microscope
  • Making mew DMEM
  • Drug selection test observation

 

Sept 6:

  • Transfection of FAPB and pEGFP-N1 into HepG2 cells in 60 mm plates
  • Maintaining cell line for HEK293FT cells

 

Sept 7:

  • Checking results for transfection on Sept 6: pEGFP-N1 showed weak GFP signal, no signals for FABP
  • Change medium for drug selection test

 

Sept 8:

  • Maintaining cell line for HEK293FT cells and HepG2 cells

 

Week 2

 

Sept 9:

  • Checking transfection for FABP construct again: Weak signals were shown from the debris of the cells
  • Transfection of AceA construct

 

Sept 11:

  • Seeding HEK293FT cells into 35mm plates for transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP
  • Cell splitting of ACE

 

Sept 12:

  • Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into HEK293FT cells

 

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

Retrieved from "http://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod1"