Protocol No. 4308 915.046 11/2001
L2, Schneider cells, Drosophila
Eppendorf Electroporation Buffer with 200 mOsmol/kg
M3 medium / 5% FCS (heat inactivated)
Eppendorf, 2 mm gap width, 400 l
RT (20-25 C)
Dr. Maren Mieth Max-Delbrueck-Centrum
fr Molekulare Medizin Robert-Rssle-Str. 10 D-13092
Berlin-Buch Phone +49 30 94062553
- Harvest the cells in the exponential growth phase and centrifuge
them (for 5 minutes, 200 x g, at room temperature).
- Resuspend the cells in M3 / 0.5% FCS, determine the number of cells
and centrifuge them (for 5 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!
- Resuspend the cells in Electroporation Buffer with 200 mOsmol/kg
(= 42% Eppendorf Hypoosmolar Electroporation Buffer + 58% Eppendorf
Isoosmolar Electroporation Buffer). When doing so, set the cell concentration
to 2 x 106 cell/ml.
- Add and mix plasmid DNA (15 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 10 minutes at room temperature.
- Carefully transfer the cell suspension from the cuvette into 3 ml
M3 / 5% FCS, and cultivate them in a 35 mm culture
dish of a 6-well plate.
Detection methods for transfection:
The expression of EGFP driven by the insect actin promotor can be detected
clearly after 24-48 hours by FACS analysis
or under a fluorescence microscope.
Transfection rate (stable):
6-9% based on the number of surviving cells.
Results were measured 24-48 hours after transfection.
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