Electroporation of A1 cells: Difference between revisions
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Ice
4 mM cuvettes, N+1 cuvettes
Steinberg's, at 4 C.
Square pulse electroporator (we use the BTX 830 Squarporator)
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
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==Materials:== | ==Materials:== | ||
<li> Ice | <li> Ice | ||
<li> 4 mM cuvettes, N+1 cuvettes | |||
<li> Steinberg's, at 4 C. | |||
<li> Square pulse electroporator (we use the BTX 830 Squarporator) | |||
1) Put cuvettes on ice, and cool down Steinberg's on ice | 1) Put cuvettes on ice, and cool down Steinberg's on ice | ||
Revision as of 07:20, 5 September 2014
Materials:
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)