( fer (= 68% Eppendorf Hypoo...)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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