Protocol No. 4308 915.021 11/1999
NIH 3T3, mouse embryo
Plasmid pEGFP-N1 (in bidistilled H2
Eppendorf Hypoosmolar Electroporation Buffer (PH)
DMEM / 10% FCS
Eppendorf, 2 mm gap width, 400 l
RT (20-25 C)
Dr. Rainer Pepperkok EMBL-Europisches
Laboratorium fr Molekulare Biologie
Meyerhofstr. 1 D-69117 Heidelberg Phone +49 6221 387-332
Fax +49 6221 387-306
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 DMEM / 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 doing
so, set the cell concentration to 1 x 10
- 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 3-5 ml DMEM / 10% FCS, and cultivate them in a 55 mm
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.
65% based on the number of surviving cells
55% based on the initial number of cells used for the experiment
Results were measured 48 hours after transfection.
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