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H73C11

fer (= 68% Eppendorf Hypoosmolar Electroporation Buffer + 32% Eppendorf Isoosmolar Electroporation buffer). When doing so, set the cell concentration to 1 x 106 cells/ml.
  • Add and mix plasmid DNA (10-20 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) 160 V Time constant (T) 40 s No. of pulses (n) 1
  • After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  • Carefully transfer the cell suspension from the cuvette to 5 ml RPMI 1640 / 10% FCS, and cultivate it in a 60 mm culture dish.
    Note: After pulsing, the cells should be incubated for 2-3 h at 37 C before any centrifugation is performed, to ensure resealing of the membrane.
  • Detection methods for transfection:
    The expression of the plasmid pEGFP-N1 can be detected clearly after 24-48 hours with the aid of FACS analysis or under a fluorescence microscope. Result: Survival rate: 250% Transfection rate: 20% based on the initial number of cells used for the experiment
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