Difference between revisions of "Electroporation of A1 cells"
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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" | ||
| + | !style="width: 150px" | 1x | ||
| + | !style="width: 100px" | FW (MW) | ||
| + | !style="width: 100px" | 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 | # 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 | ||
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# 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 | ||
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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