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721.221

Multiporator Transfection Protocol Protocol No. 4308 915.050 11/2001 Cell line 721.221, EBV transfected B cell line Transfection with pcDNA3.1/V5-His-TOPO (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI / 10% FCS (200 U/ml penicillin; 200 g/ml streptomycin, 2 mM glutamine) Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Britta Eiz-Vesper Medizinische Hochschule Hannover Institut fr Transfusionsmedizin
Feodor-Lynen-Str. 21 D-30625 Hannover
Phone +49 160-3680653 Fax +49 511-5322079 e-mail: eiz-vesper.britta@mh-hannover.de
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 300 x g, at room temperature).
  2. Resuspend the cells in RPMI / 0.5% FCS, determine the number of cells and centrifuge them (for 5-10 minutes, 300 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 2 x 107 cells/ml.
  4. Add and mix plasmid DNA (25 g/ml final concentration, in bidistilled H2O).
  5. Transfer 800 l cell suspension into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 400 V Time constant (T) 50 s No. of pulses (n) 2
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5 minutes at room temperature.
  8. Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 5 ml RPMI / 10% FCS, and cultivate them in a little culture flask.
Detection methods for transfection:
Selection and limiting dilution assay for stable transfection after 48 hours. Result: Survival rate: 85% Transfection rate: 40% based on the number of surviving cells. Results were measured 48 hours after transfection.


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