Electroporation of A1 cells: Difference between revisions
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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'' | |||
{| class="wikitable" | {| class="wikitable" | ||
!style="width: 150px" | 1x | !style="width: 150px" | 1x | ||
| Line 22: | Line 16: | ||
|8.47 g | |8.47 g | ||
|- | |- | ||
|0.67 mM KCl 74.56 1.68 ml (1M) | |0.67 mM KCl | ||
|0.44 mM Ca(NO3) 236.20 1.1 ml (1M) | |74.56 | ||
|1.3 mM MgSO4 246.47 3.25 ml (1M) | |1.68 ml (1M) | ||
|4.6 mM Tris pH 7.8-8.0 5.75 ml (2M) | |- | ||
|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 == | |||
# 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 | |||
Latest revision as of 14:25, 8 June 2015
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
- 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