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Team:Hong Kong HKUST/notebook/mod1
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</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/ | + | <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/ | + | <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/ | + | <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/ | + | <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"> | + | <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"> | + | <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"> | + | <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"> | + | <li><a href="https://2013.igem.org/Team:Hong_Kong_HKUST/safety">Safety</a></li> |
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</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/ | + | <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/ | + | <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/ | + | <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/ | + | <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> | ||
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<div> | <div> | ||
<a href='#' class='head'>Week 4</a> | <a href='#' class='head'>Week 4</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>June 25:</strong></p> | ||
| + | <ul> | ||
| + | <li>Prepared DMEM in TC room</li> | ||
| + | <li>Added FBS (Serum) & Antibiotics into DMEM</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>June 27:</strong></p> | ||
| + | <ul> | ||
| + | <li>Designed the experiment for testing FA concentration by GCMS</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>June 28:</strong></p> | ||
| + | <ul> | ||
| + | <li>HepG2 cell culturing (first generation)</li> | ||
| + | <li></li> | ||
| + | </ul><br> | ||
</div> | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
| - | <div id="satu" align="center"> <h1>July 2013</h1> | + | <div id="satu" align="center"> <h1><center>July 2013</center></h1> |
<div> | <div> | ||
<a href='#' class='head'>Week 1</a> | <a href='#' class='head'>Week 1</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>July 2:</strong></p> | ||
| + | <ul> | ||
| + | <li>HepG2 cell passaging</li> | ||
| + | <li>Made sodium palmitate from powder to solution by 50% Ethanol</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 3:</strong></p> | ||
| + | <ul> | ||
| + | <li>Used GCMS to quantify the FA in the medium</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 4:</strong></p> | ||
| + | <ul> | ||
| + | <li>Passaged HepG2 cells</li> | ||
| + | <li>Changed medium for cell line</li> | ||
| + | <li>Searched for information of MTT assay and Transfection by lipofectamine</li> | ||
| + | <li>Got GCMS results for the last day: Not accurate/usable</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 5:</strong></p> | ||
| + | <ul> | ||
| + | <li>Repeated GCMS test again testing FA in medium and we made</li> | ||
| + | <li>Got the result of GCMS: Not accurate/usable</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</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'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>July 8:</strong></p> | ||
| + | <ul> | ||
| + | <li>Passaged HepG2 cells</li> | ||
| + | <li>Used some HepG2 cells to extract genomic DNA</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 9:</strong></p> | ||
| + | <ul> | ||
| + | <li>Another try of GCMS with a gradient concentration of FA</li> | ||
| + | <li>Got the result of GCMS: Not match with the concentration we made</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 10:</strong></p> | ||
| + | <ul> | ||
| + | <li>Repeated GCMS: No accurate results</li> | ||
| + | <li>Changed medium for the cell line</li> | ||
| + | <li>Checked lipofectamine protocol</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 11:</strong></p> | ||
| + | <ul> | ||
| + | <li>GCMS again for the same sodium palmitate we made: No accurate results</li> | ||
| + | <li>Passaged HepG2 cell line</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 12:</strong></p> | ||
| + | <ul> | ||
| + | <li>Prepared reagents for MTT assay for cell viability test, protocols checked</li> | ||
| + | <li>Changed the medium of the cell line</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</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'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>July 15:</strong></p> | ||
| + | <ul> | ||
| + | <li>Seeded HepG2 cells into 96-well plate for MTT cell viability test and adding FA</li> | ||
| + | <li>Seeded HepG2 cells into 96-well plate for transfection of PEGFP-N1</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 16:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of PEGFP-N1 into HepG2 cells</li> | ||
| + | <li>Passaged HepG2 cells</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 17:</strong></p> | ||
| + | <ul> | ||
| + | <li>Changed the medium after transfection had been done in 24 hours</li> | ||
| + | <li>Did MTT assay and getting result (adding FA in the wrong time, cannot be used)</li> | ||
| + | <li>Made new DMEM</li> | ||
| + | <li>Changed medium for the cell line</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 18:</strong></p> | ||
| + | <ul> | ||
| + | <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> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 19:</strong></p> | ||
| + | <ul> | ||
| + | <li>Changed the medium for the HepG2 cell line</li> | ||
| + | <li>Seeded cells for another MTT assay</li> | ||
| + | <li>Checked HepG2 cells for transfection: growing slow, 30% confluency, cannot do transfection</li> | ||
| + | <li>Seeded cells on a 96-well plate for transfection in the coming week</li> | ||
| + | <li>Passaged HepG2 cells</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div> | <div> | ||
<a href='#' class='head'>Week 4</a> | <a href='#' class='head'>Week 4</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>July 22:</strong></p> | ||
| + | <ul> | ||
| + | <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>Coated coverslips with polylysine for HepG2 cell staining and fixation</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 23:</strong></p> | ||
| + | <ul> | ||
| + | <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>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> | ||
| + | <p> </p> | ||
| + | <p><strong>July 24:</strong></p> | ||
| + | <ul> | ||
| + | <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 changed medium of the cell line</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 25:</strong></p> | ||
| + | <ul> | ||
| + | <li>Got results for cell staining and fixation: No obvious overlapping pattern between GPF signal and Mito-tracker red.</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 26:</strong></p> | ||
| + | <ul> | ||
| + | <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 pCMV/myc/mito/GFP </li> | ||
| + | <li>Seeded HepG2 cells on two 96-well plate, for transfection of FABP construct and MTT assay in 48 hours</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div> | <div> | ||
<a href='#' class='head'>Week 5</a> | <a href='#' class='head'>Week 5</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>July 29:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of FABP construct with pEGFP-N1 as control</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> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>July 30:</strong></p> | ||
| + | <ul> | ||
| + | <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 pCMV/myc/mito/GFP</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> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| + | |||
<div id="satu" align="center"> <h1>August 2013</h1> | <div id="satu" align="center"> <h1>August 2013</h1> | ||
<div> | <div> | ||
<a href='#' class='head'>Week 1</a> | <a href='#' class='head'>Week 1</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>Aug 1:</strong></p> | ||
| + | <ul> | ||
| + | <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>Got results for MTT assay in 48 hours</li> | ||
| + | </ul><br> | ||
| + | <p><strong>Aug 2:</strong></p> | ||
| + | <ul> | ||
| + | <li>Checked if transfection (after 22 hrs) worked </li> | ||
| + | <ul> | ||
| + | <li>No GFP signal for pEFGP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | </ul> | ||
</div> | </div> | ||
</div> | </div> | ||
<div> | <div> | ||
<a href='#' class='head'>Week 2</a> | <a href='#' class='head'>Week 2</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>Aug 5: </strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of HepG2 cells on 10cm Plates </li> | ||
| + | <li>Passaged P(n+8) HepG2 cells into one P(n+9)</li> | ||
| + | <li>Seeded HepG2 cells on 96-well plates for transfection </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 6:</strong></p> | ||
| + | <ul> | ||
| + | <li>Trouble shooting for transfection of HepG2 cells on 96 well plates </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 7: </strong></p> | ||
| + | <ul> | ||
| + | <li>Observe transfected cell. GFP signal expressed </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 MTT assay </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 8:</strong></p> | ||
| + | <ul> | ||
| + | <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>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>Prepared new cell line: HEK293FT </li> | ||
| + | <li>Conducted MTT assay </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
<div> | <div> | ||
<a href='#' class='head'>Week 3</a> | <a href='#' class='head'>Week 3</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>Aug 12: </strong></p> | ||
| + | <ul> | ||
| + | <li>MTT Assay without seeding and incubation </li> | ||
| + | <li>Changed medium for HepG2 and HEK293FT cell lines </li> | ||
| + | <li>Prepared polylysine coated coverslips </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 13:</strong></p> | ||
| + | <ul> | ||
| + | <li>Seeded cells using HEK293FT cell line for transfection </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 15:</strong></p> | ||
| + | <ul> | ||
| + | <li>Cells detached and not in good condition for transfection </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 16:</strong></p> | ||
| + | <ul> | ||
| + | <li>Seeded cells HEK293FT cell line for transfection </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
<div> | <div> | ||
<a href='#' class='head'>Week 4</a> | <a href='#' class='head'>Week 4</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>Aug 19:</strong></p> | ||
| + | <ul> | ||
| + | <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> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 20:</strong></p> | ||
| + | <ul> | ||
| + | <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> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 22:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of pEGFP-N1 and FABP construct into HEK293FT cells</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> | ||
| + | <p> </p> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <div> | ||
| + | <a href='#' class='head'>Week 5</a> | ||
| + | <div class='content' align="left"> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 26:</strong></p> | ||
| + | <ul> | ||
| + | <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> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 28:</strong></p> | ||
| + | <ul> | ||
| + | <li>Changing medium of transfection of the previous day</li> | ||
| + | <li>Staining and fixation of </li> | ||
| + | <li>Drawing MTT standard curves</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 29:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into HEK293FT cells</li> | ||
| + | <li>Maintaining HEK293FT and HepG2 cell line</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 30:</strong></p> | ||
| + | <ul> | ||
| + | <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 HEK293FT cells: No obvious overlapping pattern of GFP signals and Mito-tracker</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 pCMV/myc/mito/GFP</li> | ||
| + | <li>Passaging HEK293FT cell line</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 31:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection with pEGFP-N1 and pCMV/PolyA/EGFP into HEK293FT cells</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 365: | Line 720: | ||
<div> | <div> | ||
<a href='#' class='head'>Week 1</a> | <a href='#' class='head'>Week 1</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>Sept 1:</strong></p> | ||
| + | <ul> | ||
| + | <li>Checking results for transfection on Aug 31: pEGFP-N1 had strong signal while PolyA did not have signals</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 2:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of FABP construct and pEGFP-N1 in HepG2 cells</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> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 3:</strong></p> | ||
| + | <ul> | ||
| + | <li>Adding FA into HepG2 cells been transfected by FABP construct and pEGFP-N1</li> | ||
| + | <li>Maintaining HepG2 and HEK293FT cell line</li> | ||
| + | <li>Drug selection Test on HEK293FT test by purmycin</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 4:</strong></p> | ||
| + | <ul> | ||
| + | <li>Drug selection test on HEK293FT cells by neomycin and puromycin</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 5:</strong></p> | ||
| + | <ul> | ||
| + | <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>Drug selection test observation</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 6:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of FAPB and pEGFP-N1 into HepG2 cells in 60 mm plates</li> | ||
| + | <li>Maintaining cell line for HEK293FT cells</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 7:</strong></p> | ||
| + | <ul> | ||
| + | <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> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 8:</strong></p> | ||
| + | <ul> | ||
| + | <li>Maintaining cell line for HEK293FT cells and HepG2 cells</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
<div> | <div> | ||
<a href='#' class='head'>Week 2</a> | <a href='#' class='head'>Week 2</a> | ||
| - | <div class='content'> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>Sept 9:</strong></p> | ||
| + | <ul> | ||
| + | <li>Checking transfection for FABP construct again: Weak signals were shown from the debris of the cells</li> | ||
| + | <li>Transfection of <i>AceA</i> construct</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 11:</strong></p> | ||
| + | <ul> | ||
| + | <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 ACE</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Sept 12:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of pEGFP-N1, pCMV/myc/mito/EGFP and pCMV/myc/mito/GFP into HEK293FT cells</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
<div> | <div> | ||
<a href='#' class='head'>Week 3</a> | <a href='#' class='head'>Week 3</a> | ||
| - | <div class='content'>Content 1 | + | <div class='content' align="left">Content 1 |
</div> | </div> | ||
</div> | </div> | ||
<div> | <div> | ||
<a href='#' class='head'>Week 4</a> | <a href='#' class='head'>Week 4</a> | ||
| - | <div class='content'>Content 1 | + | <div class='content' align="left">Content 1 |
</div> | </div> | ||
</div> | </div> | ||
<div> | <div> | ||
<a href='#' class='head'>Week 5</a> | <a href='#' class='head'>Week 5</a> | ||
| - | <div class='content'>Content 1 | + | <div class='content' align="left">Content 1 |
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 23:13, 27 September 2013
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
