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Multiporator Transfection Protocol Protocol No. 4308 915.034 11/1999 Cell line A 20, mouse lymphoma B-cell line Transfection with Plasmid pEGFP-N1 (in bidistilled H₂O) Electroporation buffer Eppendorf Isoosmolar Electroporation Buffer (PI) Culture medium RPMI 1640 / 10% FCS Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Hans Hcker Institut fr Med. Mikrobiologie, Immunologie und Hygiene
Technische Universitt Mnchen Trogerstr. 4a D-81675 Mnchen
Phone +49 89 4140 4184 Fax +49 89 4140 4183 e-mail: hans.haecker@lrz.tu-muenchen.de

1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 200 x g, at room temperature).
2. Resuspend the cells in RPMI 1640 / 0.5% FCS, determine the number of cells and centrifuge them (for 5-10 minutes, 200 x g, at room temperature). 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 Isoosmolar Electroporation buffer. When doing so, set the cell concentration to 3.8 x 10⁶ cells/ml.
4. Add and mix plasmid DNA (25 g/ml final concentration, in bidistilled H₂O).
5. Transfer 800 l cell suspension into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
6. Electroporation:
   
   Mode Eukaryotes Voltage (V) 600 V Time constant (T) 50 s No. of pulses (n) 2
   
7. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
8. 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.
9. 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: 250% Transfection rate: 75% based on the initial number of cells used for the experiment Results were measured 24 hours after transfection.


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