Using GFP Plasmid to Transfect Dinn-1, Myd88, and Dv-1 Cells to See Transfection Ability
Cells had already been plated here, and here in a 12 well plate. Plate layout:
| 1 | 2 | 3 | 4 | |||||
|---|---|---|---|---|---|---|---|---|
| A | Dv-1 control | Myd88 control | Dinn 3 Control | Dinn 3 Control | ||||
| B | Dv-1 transfected | Myd88 transfected | Dinn 3 transfected | Dinn 3 transfected | ||||
| C | Dv-1 transfected | Myd88 transfected | Dinn 3 transfected | Dinn 3 transfected | ||||
Using the Miris Bio Trans-IT 2020 transfection reagent and the pAc5 GFP plasmid that have been used previously. The pAc5 plasmid is already concentrated to 1ug/ul for use.
- Thawed plasmid and reagent
- Vortexed and spun them down
- All processes done in the hood
- Made 2 mixes:
- Tube 1:
- control
- 400ul of no serum schneider’s medium
- Tube 2:
- reagent
- 800ul of no serum schneider’s medium
- 8ul concentrated plasmid
- 8ul of 2020 reagent
- pipette mixed gently
- Incubated tube 2 for 30 min in the hood for complexes to form
- Then, added reagents dropwise to their respective wells (see layout above)
- 100ul of control to control wells
- 102ul of reagent to reagent wells
- Rocked plate back and forth for a minute
- Placed plate in the 23 incubator for overnight
This plate was looked at every 24 hours for 5 days in Dr. Ackley’s scope to look for GFP presence in the cells. Day 5 was the best imaged because each cell type had GFP positive cells. All images can be found here