Electroporation of A1 cells: Difference between revisions
Jump to navigation
Jump to search
Ice
4 mM cuvettes, N+1 cuvettes
Steinberg's, at 4 C.
Square pulse electroporator (we use the BTX 830 Squarporator)
No edit summary |
No edit summary |
||
| Line 11: | Line 11: | ||
# Electroporate cells in following conditions and put back on ice: Myoblasts: 100V, 35 msec, 5 pulses, Myotubes: 75 V, 35 msec, 5pulses | # Electroporate cells in following conditions and put back on ice: Myoblasts: 100V, 35 msec, 5 pulses, Myotubes: 75 V, 35 msec, 5pulses | ||
# Plate cells as normal | # Plate cells as normal | ||
==Steinberg's== | ==Steinberg's== | ||
{| class="wikitable" | |||
!style="width: 300px" | 1x | |||
!style="width: 200px" | FW (MW) | |||
!style="width: 200px" | 5x/500 ml | |||
|- | |||
| | |||
58 mM NaCl 58.44 8.47 g | |||
0.67 mM KCl 74.56 1.68 ml (1M) | 0.67 mM KCl 74.56 1.68 ml (1M) | ||
0.44 mM Ca(NO3) 236.20 1.1 ml (1M) | 0.44 mM Ca(NO3) 236.20 1.1 ml (1M) | ||
1.3 mM MgSO4 246.47 3.25 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) | 4.6 mM Tris pH 7.8-8.0 5.75 ml (2M) | ||
Revision as of 08:12, 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: Myoblasts: 100V, 35 msec, 5 pulses, Myotubes: 75 V, 35 msec, 5pulses
- Plate cells as normal
Steinberg's
| 1x | FW (MW) | 5x/500 ml |
|---|---|---|
|
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) |