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Embryonic stem cells, mouse

30 minutes to guarantee a successful electroporation!

  • Resuspend the cells at a concentration of 1 x 107 cells/ml in Hypoosmolar Electroporation Buffer.
  • Mix 750 l cell suspension with 50 l DNA solution (1 g/l in Isoosmolar Electroporation Buffer).
  • Transfer 800 l cell/DNA solution into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
  • Electroporation:

    Mode Eukaryotes Voltage (V) 300 V Time constant (T) 100 s No. of pulses (n) 1
  • After the pulse, let the cells shortly recover for 5 minutes at room temperature and then carefully transfer the cells to 10 ml ES-medium.
  • Plate the cells onto 100 mm culture dishes prepared with inactivated mouse embryonic fibroblasts feeder layers at a density of 1-1.5 x 106 cells per plate.
  • The next day change medium and start selection 24 hours after the electroporation with 250 g/ml medium of G418. Change selection medium every day.
  • 7-8 days after the electroporation pick the surviving individual ES-cell colonies under a stereo microscope, culture and expand them individually. Take only colonies with sharp borders and proper morphology.
  • After expansion freeze down at least one aliquot of each clone and use another for DNA preparation to identify correctly transfected clones
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