Team:Hong Kong HKUST/notebook/mod1

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<ol class="pos_fixed">
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<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>

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

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