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B16

antee a successful electroporation!

  • Resuspend the cells in Hypoosmolar Electroporation Buffer. Add and mix plasmid DNA (20 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) 400 V Time constant (T) 100 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 DMEM /10% FCS, and cultivate them in a 55 mm culture dish.
    • Detection methods for transfection:
    The expression of the plasmid pEGFP-N1 by viable B16 cells can be detected after 24-48 hours with a flow cytometer by costaining with propidium iodide (final concentration 1 g/ml). Incubate for 10 min. at RT. Result: Survival rate: 68.91% Transfection rate: 78.21% based on the number of surviving cells. Results were measured 24 hours after transfection.


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