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Team:Hong Kong HKUST/notebook/mod1
From 2013.igem.org
(Difference between revisions)
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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"> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>Aug 12: </strong></p> | ||
| + | <ul> | ||
| + | <li>MTT Assay without seeding ad incubation </li> | ||
| + | <li>Changed medium for HepG2 and HEK 293 FT cell lines </li> | ||
| + | <li>Prepared polylysine coated coverslips </li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 13:</strong></p> | ||
| + | <ul> | ||
| + | <li>Seeded cells using HEK 293 FT 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 HEK 293 FT 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' align="left"> | + | <div class='content' align="left"> |
| + | <p> </p> | ||
| + | <p><strong>Aug 19:</strong></p> | ||
| + | <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> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 20:</strong></p> | ||
| + | <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> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 22:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection of PEGFP-N1 and FABP into HEK 293FT 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> | ||
| + | </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 PCMY/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 PCMY/myc/mito/GFP into HEK293FT cells</li> | ||
| + | <li>Maintaining HEK 293FT 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 PCMY/myc/mito/GFP</li> | ||
| + | <li>Seeing the results for transfected HEK 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 HEK 293FT cells for transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP</li> | ||
| + | <li>Passaging HEK cell line</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
| + | <p><strong>Aug 31:</strong></p> | ||
| + | <ul> | ||
| + | <li>Transfection with PEGFP-N1 and PCMV/PolyA/EGFP into HEK 293FT cells</li> | ||
| + | </ul> | ||
| + | <p> </p> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 16:07, 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
August 2013
Week 1
Aug 1:
- Seeded cells for transfection of PEGFP-N1, FABP, PCMV/myc/mito/EGFP and PCMY/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/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/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: HEK 293 FT
- Conducted MTT assay
Week 3
Aug 12:
- MTT Assay without seeding ad incubation
- Changed medium for HepG2 and HEK 293 FT cell lines
- Prepared polylysine coated coverslips
Aug 13:
- Seeded cells using HEK 293 FT cell line for transfection
Aug 15:
- Cells detached and not in good condition for transfection
Aug 16:
- Seeded cells HEK 293 FT cell line for transfection
Week 4
Aug 19:
- 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.
Aug 20:
- 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
Aug 22:
- Transfection of PEGFP-N1 and FABP into HEK 293FT cells
- Changing medium for HEK 293FT 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 PCMY/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 PCMY/myc/mito/GFP into HEK293FT cells
- Maintaining HEK 293FT and HepG2 cell line
Aug 30:
- Staining and fixation for HEK cells transfected by PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP
- Seeing the results for transfected HEK cells: No obvious overlapping pattern of GFP signals and Mito-tracker
- Seeding HepG2 cells for Transfection of FABP and PEGFP-N1 in 96-well plate
- Seeding HEK 293FT cells for transfection of PEGFP-N1, PCMV/myc/mito/EGFP and PCMY/myc/mito/GFP
- Passaging HEK cell line
Aug 31:
- Transfection with PEGFP-N1 and PCMV/PolyA/EGFP into HEK 293FT cells
