Using Mirus Bio 2020 Reagent to Transfect pAc5 GFP Expression Plasmid into S2, Dv-1, and Myd88 Cells

Using the TransIT 2020 Transfection Reagent to test if I can get Myd88 cells to express GFP. This is basically a test of the transfection method on cells like might be more “primary-like.” The Dv-1 and S2 cells will be controls.

The cells were plated to 1.5*10^5 cells/mL the day before.

Plate layout:

PLATE 8 1   2   3   4  
A S2 control   Myd88 control   Myd88 control   Dv-1 control  
                 
                 
B S2 2020 1ul reagent   Myd88 1ul 2020 reagent   Myd88 3ul 2020 reagent   DV-1 1ul 2020 reagent  
                 
                 
C S2 2020 1ul reagent   Myd88 1ul 2020 reagent   Myd88 3ul 2020 reagent   DV-1 1ul 2020 reagent  
                 
                 

pAc5 concentration

  • I concentrated the last of my plasmid extraction to 1ug/ul to use for the transfection
  • Tube 6 has 124ul of 92.7ng/ul DNA. That is total 11,494ng DNA
  • I bead cleaned this extraction and eluted it in 11ul of molecular grade water, then added it to the small amount of concentrated pAc5 DNA I had previously

Transfection

  • All processes were done in the cell culture hood
  • The DNA and transfection reagent were thawed, vortexed, spun down, and kept at room temp
  • Made 3 master mix tubes with the transfection reagents:
  • Tube 1 - controls
    • 400ul Schneider’s medium
  • Tube 2 - 1ul reagent per well
    • 600ul Schneider’s medium
    • 6ul concentrated pAc5 plasmid
    • 6ul 2020 reagent
  • Tube 3 - 3ul reagent per well
    • 200ul Schneider’s medium
    • 2ul concentrated pAc5 plasmid
    • 6ul 2020 reagent
  • All tubes were pipette mixed gently
  • Tubes were left in the cell culture hood for 30 minutes so the liposome complexes could form
  • Then, the reagent mix was added dropwise to the appropriate well:
    • Tube 1: 100ul into wells A1, A2, A3, and A4
    • Tube 2: 102ul into wells B1, C1, B2, C2, B4, and C4
    • Tube 3: 104ul into wells B3, and C3
  • The plate was gently rocked back and forth a few times to try to mix the reagent around in the wells
  • Then the plate was placed in the 23C incubator and will be checked every 24 hours

Every ~24 hours images will be taken with the GFP filter and can be seen here and here