Team:Hong Kong HKUST/notebook/mod1

From 2013.igem.org

(Difference between revisions)
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<div>
<div>
       <a href='#' class='head'>Week 1</a>
       <a href='#' class='head'>Week 1</a>
-
       <div class='content' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</p>
 +
<p><strong>Aug 1:</strong></p>
 +
<ul>
 +
  <li>Seeded cells for transfection of PEGFP-N1, FABP, PCMV/myc/mito/EGFP and PCMY/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>&nbsp;</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' align="left">Content 1
+
       <div class='content' align="left">
 +
<p>&nbsp;</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>&nbsp;</p>
 +
<p><strong>Aug 6:</strong></p>
 +
<ul>
 +
  <li>Trouble shooting for  transfection of HepG2 cells on 96 well plates </li>
 +
</ul>
 +
<p>&nbsp;</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/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>&nbsp;</p>
 +
<p><strong>Aug 8:</strong></p>
 +
<ul>
 +
  <li>Observed transfection of PEGFP-N1, and (FABP/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: HEK 293 FT </li>
 +
  <li>Conducted MTT assay </li>
 +
</ul>
 +
<p>&nbsp;</p>
       </div>
       </div>
     </div>
     </div>

Revision as of 16:03, 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
Content 1
Week 4
Content 1

September 2013

Week 1
Content 1
Week 2
Content 1
Week 3
Content 1
Week 4
Content 1
Week 5
Content 1

Retrieved from "http://2013.igem.org/Team:Hong_Kong_HKUST/notebook/mod1"