Electroporation of A1 cells

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Material

  • Ice
  • 4 mM cuvettes, N+1 cuvettes
  • Steinberg's, at 4 C.
  • Square pulse electroporator (we use the BTX 830 Squarporator)


Steinberg's

1x FW (MW) 5x/500 ml
58 mM NaCl 58.44 8.47 g
0.67 mM KCl 74.56 1.68 ml (1M)
0.44 mM Ca(NO3) 236.20 1.1 ml (1M)
1.3 mM MgSO4 246.47 3.25 ml (1M)
4.6 mM Tris pH 7.8-8.0 5.75 ml (2M)


Procedure

  1. Put cuvettes on ice, and cool down Steinberg's on ice
  2. Trypsinize cells as normal, stop reaction with high serum media and spin down at 1000 rpm, 3 minutes
  3. Wash pellet with Steinberg's solution and resuspend in 300 µl of Steinberg's, keep on ice
  4. Before electroporation, make sure to electroporate a cuvette with no cells to discharge device
  5. Electroporate cells in following conditions and put back on ice: Myoblasts: 100V, 35 msec, 5 pulses, Myotubes: 75 V, 35 msec, 5pulses
  6. Plate cells as normal