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U-266

Multiporator Transfection Protocol Protocol No. 4308 915.040 11/2000 Cell line U-266, human myeloma Transfection with Plasmid pEGFP-C1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium IMDM / 10 % FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Frau Pfeifer Universittsklinikum Leipzig Molekulare Immunologie Johannisallee 30
D-04103 Leipzig Phone: +49 341 9725494 Fax: +49 341 9725489
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 200 x g, at room temperature).
  2. Resuspend the cells in IMDM / 0.5% FCS, determine the number of cells and centrifuge them (for 5-10 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 Hypoosmolar Electroporation Buffer. When doing so, set the cell concentration to 1 x 106 cells/ml
  4. Add and mix plasmid DNA (10 -20 l/ml final concentration, in bidistilled H2O) The cell suspension must be free of air bubbles.
  5. Electroporation:

    Mode Eukaryotes Voltage (V) 600 V Time constant (T) 60 s No. of pulses (n) 1
  6. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  7. Carefully transfer the cell suspension from the cuvette into 3-5 ml IMDM / 10% FCS, and cultivate them in a 55 mm culture dish.
Detection methods for transfection:
The expression of the plasmid pEGFP-C1 can be detected clearly after 24-48 hours with the aid of FACS analysis or under a fluorescence microscope. Result: Survival rate: 62% Transfection rate: 23% based on the number of surviving cells. Results were measured 48 hours after transfection.


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