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NIH 3T3

Multiporator Transfection Protocol Protocol No. 4308 915.021 11/1999 Cell line NIH 3T3, mouse embryo Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium DMEM / 10% FCS Cuvette Eppendorf, 2 mm gap width, 400 l Temperature RT (20-25 C) Reference Dr. Rainer Pepperkok EMBL-Europisches Laboratorium fr Molekulare Biologie
Meyerhofstr. 1 D-69117 Heidelberg Phone +49 6221 387-332 Fax +49 6221 387-306
e-mail: pepperko@embl-heidelberg.de
  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 DMEM / 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 10
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