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Multiporator Transfection Protocol Protocol No. 4308 915.031 12/1999 Cell line CHO, Chinese hamster ovary, Transfection with Rat-DPP IV (in bidistilled H2O), stable transfection Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium Alpha-Medium / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Sabine Grams Institut fr Molekularbiologie und Biochemie Arnimallee 22
D-14195 Berlin Phone +49 30 8445 1543 Fax +49 30 8445 1541
  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 Alpha-Medium / 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< /sup> cells/ml.
  4. Add and mix plasmid DNA (5-10 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) 290 V Time constant (T) 40 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 5 ml Alpha-Medium / 10% FCS, centrifuge it for
    5-10 minutes, 200 x g, at room temperature. Remove supernatant.
  9. Resuspend the cells in 2 ml Alpha-Medium / 10% FCS, and cultivate them in a 6 well microtiterplate.

Detection methods for transfection:

FACS method. Result: Survival rate: 98% (measured 48 hours after transfection) Transfection rate (stable): 50% based on the initial number of cells used for the experiment (after 10 days of incubation
in selection medium)


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