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Team:Hong Kong HKUST/notebook/mod1
From 2013.igem.org
(Difference between revisions)
| Line 397: | Line 397: | ||
<div> | <div> | ||
<a href='#' class='head'>Week 3</a> | <a href='#' class='head'>Week 3</a> | ||
| - | <div class='content' align="left"> | + | <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 PCMY/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> | ||
| Line 403: | Line 439: | ||
<div> | <div> | ||
<a href='#' class='head'>Week 4</a> | <a href='#' class='head'>Week 4</a> | ||
| - | <div class='content' align="left"> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>July 22:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/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 PCMY/myc/mito/GFP</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> | ||
| + | </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 PCMY/myc/mito/GFP in 24-well plate</li> | ||
| + | <li>Passaged HepG2 cells and changing 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 PCMY/myc/mito/GFP </li> | ||
| + | <li>Seeded HepG2 cells on two 96-well plate, for transfection of FABP and MTT assay in 48 hours</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
| Line 409: | Line 478: | ||
<div> | <div> | ||
<a href='#' class='head'>Week 5</a> | <a href='#' class='head'>Week 5</a> | ||
| - | <div class='content' align="left"> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>July 29:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of FABP 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>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 and seeing the results: Both PEGFP-N1 and FABP 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>Did staining and fixation of the coverslips and seeing 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> | ||
Revision as of 15:51, 19 September 2013
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: Not 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 PCMY/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 PCMY/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 PCMY/myc/mito/GFP
- Finished MTT assay to get cell viability in different FA concentrations in 24 hours
- Transferredcells containing PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/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 PCMY/myc/mito/GFP in 24-well plate
- Passaged HepG2 cells and changing 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 PCMY/myc/mito/GFP
- Seeded HepG2 cells on two 96-well plate, for transfection of FABP and MTT assay in 48 hours
Week 5
July 29:
- Transfection of FABP with PEGFP-N1 as control
- Transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/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 and seeing the results: Both PEGFP-N1 and FABP did not show GFP signals
- Changed medium after transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP
- Did staining and fixation of the coverslips and seeing the results: No obvious overlapping patterns of GFP signals and Mito-tracker red
- Passaged HepG2 cells
