Second Attempt at pAc5 GFP Plasmid Transfection with DinnDiNV 3.1 and S2 Cells

The previous attempt at a transfection with the DinnDiNV cells showed inconclusive results, there may have been some GFP expression but it was very hard to tell. I thought that maybe the 20% FBS medium could have interfierred with the transfection reagent, I had only ever used it with 10% FBS before. So I am re-doing the experiment with 3.1 cells plated in 10% FBS, 4% mushroom Schneider’s medium. Those cells settled on the plate for about 3 days until transfection. The day before transfection, I also plated S2 cells for control at about 150,000 cells per mL.

Plate layout for the transfection:

  1   2   3   4  
A S2 control   3.1 DinnDiNV control, 100ul schneider’s medium   3.1 DinnDiNV control, 100ul schneider’s medium   X  
                 
                 
B S2 2020 1ul reagent   3.1 DinnDiNV, 1ul 2020 reagent, 1ul pAc5   3.1 DinnDiNV, 1ul 2020 reagent, 1ul pAc5   X  
                 
                 
C S2 2020 1ul reagent   3.1 DinnDiNV, 1ul 2020 reagent, 1ul pAc5   3.1 DinnDiNV, 1ul 2020 reagent, 1ul pAc5   X  
                 
                 

The pAc5 plasmid was concentrated to 1ug/ul in the previous transfection, and I used an aliquot from that.

Transfection

  • All processes were done in the cell culture hood
  • The plasmid DNA and transfection reagent were thawed, vortexed, spun down, and kept at room temp
  • Made 2 master mix tubes with the transfection reagents:
  • Tube 1 - controls
    • 300ul Schneider’s medium
  • Tube 2 - 1ul reagent per well
    • 600ul Schneider’s medium
    • 6ul 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, and A3
    • Tube 2: 102ul into wells B1, C1, B2, C2, B3, and C3
      • Note that C3 had a lot less fluid added to it, I was short on reagent. It may have been that I added 106ul instead of 102ul
  • 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 in bulk here