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Transfection
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Materials required

 

  1. 35 mm plates with cells
  2. Opti- MEM with & without 20% FBS
  3. Transfection reagent
  4. Microfuge tubes (1.5 ml) & microfuge tube stand
  5. Pipettes & Micro Pipettes
  6. Suction pump
  7. CO2 incubator
     

Procedure

 

 

  1. Take 50 µl of Opti- MEM media in two fresh vials (microfuge tubes) labeled as ‘T’ & ‘C’.
  2. Add 4 µl of plasmid DNA (with Gene of interest) into the vial ‘T’.
  3. Similarly, add 4 µl of plasmid DNA (without Gene of interest) into the vial ‘C’.
  4. Add 100µl of Opti- MEM media and 16 µl of transfection reagent into a fresh vial. Mix the solution well.
  5. Incubate the vial for 5 minutes at room temperature.
  6. After incubation, the mixture is equally added into the vials ‘T’ & ‘C ’.
  7. Incubate both vials for 20 minutes at room temperature.
  8. Take two culture plates with HEK cells from the  incubator.
  9. 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%).
  10. Remove the media from cultures using the suction pump and add 2 ml of Opti- MEM to both the plates.
  11. Pour the mixture from vials T and C to the culture plates (labeled as ‘T’ & ‘C’) respectively. Mix the solution well.
  12. Incubate the plates at 370C for 4 hours in the CO2 incubator.
  13. Remove the media from culture plates using the suction pump and add 2 ml of DMEM with 20% FBS to both the plates.
  14. Incubate the plate at 370C for 24 hours in the CO2 incubator.
  15. After incubation, observe the cells under inverted fluorescence microscope and confirm transfection process using different fluorescence filters.
  16. Transfer the plate into the tissue culture bottle and store at 4°C in cold room.


 

 

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