- 35 mm plates with cells
- Opti- MEM with & without 20% FBS
- Transfection reagent
- Microfuge tubes (1.5 ml) & microfuge tube stand
- Pipettes & Micro Pipettes
- Suction pump
- CO2 incubator
- Take 50 µl of Opti- MEM media in two fresh vials (microfuge tubes) labeled as ‘T’ & ‘C’.
- Add 4 µl of plasmid DNA (with Gene of interest) into the vial ‘T’.
- Similarly, add 4 µl of plasmid DNA (without Gene of interest) into the vial ‘C’.
- Add 100µl of Opti- MEM media and 16 µl of transfection reagent into a fresh vial. Mix the solution well.
- Incubate the vial for 5 minutes at room temperature.
- After incubation, the mixture is equally added into the vials ‘T’ & ‘C ’.
- Incubate both vials for 20 minutes at room temperature.
- Take two culture plates with HEK cells from the incubator.
- Check the confluency of the cultures under an inverted fluorescence microscope. (Confluency should be 80-90%. Other ways incubate the cells till it reaches 80- 90%).
- Remove the media from cultures using the suction pump and add 2 ml of Opti- MEM to both the plates.
- Pour the mixture from vials T and C to the culture plates (labeled as ‘T’ & ‘C’) respectively. Mix the solution well.
- Incubate the plates at 370C for 4 hours in the CO2 incubator.
- Remove the media from culture plates using the suction pump and add 2 ml of DMEM with 20% FBS to both the plates.
- Incubate the plate at 370C for 24 hours in the CO2 incubator.
- After incubation, observe the cells under inverted fluorescence microscope and confirm transfection process using different fluorescence filters.
- Transfer the plate into the tissue culture bottle and store at 4°C in cold room.
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