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721.174

electroporation!

  • Resuspend the cells in Hypoosmolar Electroporation Buffer. Add and mix plasmid DNA (20 g/ml final concentration, in bidistilled H2O).
  • Add and mix plasmid DNA (12.5 g/ml final concentration, in bidistilled H2O).
  • Transfer 400 l cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of air bubbles.
  • Electroporation:

    Mode Eukaryotes Voltage (V) 200 V Time constant (T) 40 s No. of pulses (n) 1
  • After the pulse, allow the cell suspension to stand in the cuvette for 15 minutes at room temperature.
  • Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 3 ml RPMI /10% FCS, and cultivate them in a 55 mm culture dish.
  • Detection methods for transfection:
    The expression of the plasmid pEGFP-N1 by viable 721.174 cells can be detected after 24 hours with a flow cytometer by costaining with propidium iodide (final concentration 1 g/ml). Incubate for 10 min. at RT. Result: Survival rate: 72.54% Transfection rate: 20.77% based on the number of surviving cells.
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