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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| Line 28: | Line 28: | ||
|0.44 mM Ca(NO3) | |0.44 mM Ca(NO3) | ||
|236.20 | |236.20 | ||
|1.1 ml (1M) | |||
|- | |- | ||
|1.3 mM MgSO4 | |||
|1.3 mM MgSO4 246.47 3.25 ml (1M) | |246.47 | ||
|4.6 mM Tris pH 7.8-8.0 5.75 ml (2M) | |3.25 ml (1M) | ||
|- | |||
|4.6 mM | |||
|Tris pH 7.8-8.0 | |||
|5.75 ml (2M) | |||
Revision as of 08:43, 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 |
|---|---|---|
| 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) |