Protocol No. 4308 915.024 11/2001
H73C11, mouse-human, heteromyeloma (suspension cell
Plasmid pEGFP-N1 (in bidistilled H2
Eppendorf Electroporation Buffer with 150 mOsmol/kg
RPMI 1640 / 10% FCS
Eppendorf, 2 mm gap width, 400
RT (20-25 C)
Prof. Ulrich Zimmermann Lehrstuhl
fr Biotechnologie Biozentrum Universitt Wrzburg
Am Hubland D-97074 Wrzburg Phone +49 931 888 4508
Fax +49 931 888 4509
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 150 mOsmol/kg Electroporation Buffer (= 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
- Transfer 400 l cell suspension into electroporation cuvettes (2 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 / 10% FCS, and cultivate it in a 60 mm
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.
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.
20% based on the initial number of cells used for the experiment
Results were measured 48 hours after transfection.
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