Protocol No. 4308 915.042 11/2001
B16, mouse melanoma cells
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. Kirsten Falk and Dr. Olaf Rtzschke
Max Delbrck Center for Molecular Medicine Robert-Rssle-Strasse
10 D-13125 Berlin-Buch
Detection methods for transfection:
- Harvest the cells in the exponential growth phase with 0.05% trypsin-EDTA
for 1 min, stop by adding DMEM and centrifuge them (for 5 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 minutes, 200 x g, at room temperature). Remove
- Resuspend the cells in medium, set the cell number to 1 x 106
cells/ml and centrifuge them (for 5 minutes, 200 x g, at room temperature).
Note: The overall incubation time in the Eppendorf Electroporation
Buffer must not exceed 30 minutes to guar
antee a successful electroporation!
- Resuspend the cells in Hypoosmolar Electroporation Buffer. Add and
mix plasmid DNA (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.
Time constant (T)
No. of pulses (n)
- After the pulse, allow the cell suspension to stand in the cuvette
for 15 minutes at room temperature.
- Carefully transfer the cell suspension with a pasteur pipette from
the cuvette into 3 ml DMEM /10% FCS, and cultivate them in a 55 mm culture
The expression of the plasmid pEGFP-N1 by viable B16 cells can be detected after 24-48 hours with a flow cytometer by
costaining with propidium iodide (final concentration 1 g/ml). Incubate for 10 min. at RT.
78.21% based on the number of surviving cells.
Results were measured 24 hours after transfection.
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