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)
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# Put cuvettes on ice, and cool down Steinberg's on ice | # Put cuvettes on ice, and cool down Steinberg's on ice | ||
# Trypsinize cells as normal, stop reaction with high serum media and spin down at 1000 rpm, 3 minutes | # Trypsinize cells as normal, stop reaction with high serum media and spin down at 1000 rpm, 3 minutes | ||
# Wash pellet with Steinberg's solution and resuspend in 300 µl of Steinberg's, keep on ice | # Wash pellet with Steinberg's solution and resuspend in 300 µl of Steinberg's, keep on ice | ||
# Before electroporation, make sure to electroporate a cuvette with no cells to discharge device | # Before electroporation, make sure to electroporate a cuvette with no cells to discharge device | ||
# Electroporate cells in following conditions and put back on ice: | # Electroporate cells in following conditions and put back on ice: | ||
Myoblasts: 100V, 35 msec, 5 pulses | Myoblasts: 100V, 35 msec, 5 pulses | ||
Myotubes 75 V, 35 msec, 5pulses | Myotubes 75 V, 35 msec, 5pulses | ||
# Plate cells as normal | # Plate cells as normal | ||
Revision as of 07:23, 5 September 2014
Materials:
- Put cuvettes on ice, and cool down Steinberg's on ice
- Trypsinize cells as normal, stop reaction with high serum media and spin down at 1000 rpm, 3 minutes
- Wash pellet with Steinberg's solution and resuspend in 300 µl of Steinberg's, keep on ice
- Before electroporation, make sure to electroporate a cuvette with no cells to discharge device
- Electroporate cells in following conditions and put back on ice:
- Plate cells as normal
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)