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Team:Hong Kong HKUST/notebook/mod1
From 2013.igem.org
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Revision as of 22:37, 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
