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Embryonic stem cells, mouse

Multiporator Transfection Protocol Protocol No. 4308 915.039 12/1999 Cell line Embryonic stem cells (ES cells), mouse, R1. Passage 15 Transfection with Linearized targeting vector (12 kb) with neoR-selection marker Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium DMEM / 15% FCS, 2 mM glutamine, 1 mM pyruvate, 1 x MEM, 1 x nucleoside mix, 1 x Pen/Strep, 1000 U LIF Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Michael Bsl ZMNH Transgene Technologie Martinistr. 42 D-20246 Hamburg
Phone +49 40 42803 6663 Fax +49 40 42803 6659 e-mail: boesl@plexus.uke.uni-hamburg.de
  1. Harvest the cells 36-40 hours after the last passage, determine the number of cells and centrifuge the cells (5 minutes, 200 x g, at room temperature).
  2. Wash the cells in Hypoosmolar Electroporation Buffer: Resuspend the cells in 10 ml Hypoosmolar Buffer and collect them by centrifugation (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
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