Using Mirus Bio 2020 Reagent to Transfect pAc5 GFP Expression Plasmid into S2 and DinnDiNV Cells

Using the TransIT 2020 Transfection Reagent to test if I can get the DinnDiNV cells to express GFP. I will be testing both 1ul of 2020 reagent, and 3ul 2020 reagent. Both of these volumes have worked for other cell types.

The cells were plated the day before/the week before here, and here.

Plate layout:

PLATE 8 1   2   3   4  
A S2 control   3.0 top fluid, strained   3.0 bottom fluid, strained   3.1 top fluid, strained  
                 
                 
B S2 1ul 2020 reagent   3.0 top fluid, strained 1ul 2020 reagent   3.0 bottom fluid, strained 3ul 2020 reagent   3.1 top fluid, strained 1ul 2020 reagent  
                 
                 
C S2 1ul 2020 reagent   3.0 top fluid, strained 1ul 2020 reagent   3.0 bottom fluid, strained 3ul 2020 reagent   3.1 top fluid, strained 1ul 2020 reagent  
                 
                 

pAc5 concentration 20230502

  • Latest pAc5 extraction was done here
  • There wasn’t that great yields, but I can combine some of the extractions and concentrate them down into ~9ug aliquiots
  • Combine tubes 1 and 2:
    • 100ul * 51.4ng/ul = 5,140ng
    • 100ul * 47.1ng/ul = 4,710ng
    • total: 9,850ng
  • Combine tubes 3 and 4:
    • 100ul * 50.2ng/ul = 5,020ng
    • 100ul * 47.6ng/ul = 4,760ng
    • total: 9,780ng
  • Combine tubes 5, 6, and 7:
    • 100ul * 43ng/ul = 4,300ng
    • 100ul * 44.7ng/ul = 4,470ng
    • 100ul * 12.1ng/ul = 1,200ng
    • total: 9,970ng
  • Concentrated with 1X bead clean up and elute in 30ul
  • Concentrated with a second 1X bead clean up and eluted in 10ul of 10mM Tris HCl, so that there are about 10ug in 10ul , or 1ug/ul concentration

Transfection 20230510

  • 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 in bulk here