Protocol No. 4308 915.032 03/2000
BW 5147, mouse lymphoma T-cell line
Plasmid pEGFP-N1 (in bidistilled H2
Eppendorf Hypoosmolar Electroporation Buffer (PH)
RPMI 1640 / 10% FCS
Eppendorf, 4 mm gap width, 800 l
RT (20-25 C)
Dr. Hans Hcker Institut fr
Med. Mikrobiologie, Immunologie und Hygiene
Technische Universitt Mnchen Trogerstr. 4a
Phone +49 89 4140 4184 Fax +49 89 4140 4183 e-mail:
Detection methods for transfection:
- Harvest the cells in the exponential growth phase and centrifuge them
(for 5-10 minutes, 200 x g, at room temperature).
- 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).
Note: The overall incubation time in the Eppendorf Electroporation
Buffer must not exceed 30 minutes to guarantee a successful electroporation!
- Resuspend the cells in Hypoosmolar Electroporation Buffer. When
so, set the cell concentration to 5 x 106 cells/ml.
- Add and mix plasmid DNA (25 g/ml final concentration, in bidistilled
- Transfer 800 l cell suspension into electroporation cuvettes (4 mm
gap width). The cell suspension must be free of air bubbles.
Time constant (T)
No. of pulses (n)
- 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 1 ml RPMI 1640 / 10% FCS, and cultivate them in a 35 mm culture dish.
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.
84% based on the number of surviving cells
120% based on the initial number of cells used for the experiment
Results were measured 48 hours after transfection.
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