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li>Resuspend the cells in Electroporation Buffer with 200 mOsmol/kg (= 42% Eppendorf Hypoosmolar Electroporation Buffer + 58% Eppendorf Isoosmolar Electroporation Buffer). When doing so, set the cell concentration to 1 x 106 cells/ml.
  • 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) 670 V Time constant (T) 100 s No. of pulses (n) 1
  • After the pulse, allow the cell suspension to stand in the cuvette for 10 minutes at room temperature.
  • Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 3 ml RPMI / 10% FCS, and cultivate it in a 55 mm culture dish.
  • Detection methods for transfection:
    The expression of the plasmid pcDNA3-EGFP can be detected clearly after 24-48 hours under a fluorescence microscope. Result: Survival rate: > 95% Transfection rate: 70% based on the initial number of cells used for the experiment Results were measured 24 hours after transfection
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