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Multiporator Transfection Protocol Protocol No. 4308 915.046 11/2001 Cell line L2, Schneider cells, Drosophila Transfection with pActin-SV-EGFP Electroporation buffer Eppendorf Electroporation Buffer with 200 mOsmol/kg Culture medium M3 medium / 5% FCS (heat inactivated) Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Maren Mieth Max-Delbrueck-Centrum fr Molekulare Medizin Robert-Rssle-Str. 10 D-13092 Berlin-Buch Phone +49 30 94062553
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5 minutes, 200 x g, at room temperature).
  2. 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!

  3. 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 c oncentration to 2 x 106 cell/ml.
  4. Add and mix plasmid DNA (15 g/ml final concentration, in bidistilled H2O).
  5. Transfer 400 l cell suspension into electroporation cuvettes (2 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 180 V Time constant (T) 100 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 10 minutes at room temperature.
  8. 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. Result: Survival rate: 70-80% Transfection rate (stable): 6-9% based on the number of surviving cells. Results were measured 24-48 hours after transfection.



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