( ing so set the cell conce...)EL 4

ing so, set the cell concentration to 3.8 x 10⁶ cells/ml.

Add and mix plasmid DNA (25 g/ml final concentration, in bidistilled H₂O).

Transfer 800 l cell suspension into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.

Electroporation:

Mode Eukaryotes Voltage (V) 480 V Time constant (T) 50 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 / 0.5% FCS, and centrifuge it for 5-10 minutes, 200 x g, at room temperature. Remove supernatant.

Resuspend the cells in 3 ml RPMI 1640 / 10% FCS, and cultivate them in a 55 mm culture dish.

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: 81% Transfection rate: 40% based on the number of surviving cells
32% based on the initial number of cells used for the experiment Results were measured 48 hours after transfection.


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