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Multiporator Transfection Protocol Protocol No. 4308 915.022 11/1999 Cell line COS-7, monkey, kidney Transfection with Plasmid RSV/lacZ (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium DMEM / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature 4 C Reference Dr. Helena Edlund Department of Microbiology University of Umea Umea Sweden
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  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 200 x g, at 4 C).
  2. Resuspend the cells in DMEM / 0.5% FCS, determine the number of cells and centrifuge them (for 5-10 minutes, 200 x g, at 4 C). Remove supernatant.

    Note: The overall incubation time in the Eppendorf Electroporation Buffer must not exceed 30 minutes to guarantee a successful electroporation!

  3. Resuspend the cells in Hypoosmolar Electroporation Buffer. When doing so, set the cell concentration to 1 x 106 cells/ml.
  4. Add and mix plasmid DNA (5 g/ml final concentration, in bidistilled H2O).
  5. Transfer 400 l cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 600 V Time constant (T) 40 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5 minutes on ice.
  8. Carefully transfer the cell suspension from the cuvette to 3-5 ml DMEM / 10% FCS, and cultivate them in a 55 mm culture dish.
Detection methods for transfection:
The expression of the plasmid RSV/lacZ can be detected after 48 hours with X-gal-staining. Result: Survival rate: n.d. Transfection rate: 30-40% based on the initial number of cells used for the experiment Results were measured 48 hours after transfection.



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