Electroporation of A1 cells: Difference between revisions

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==Materials:==
== Material ==
<li> Ice
* Ice
<li> 4 mM cuvettes,  N+1 cuvettes
* 4 mM cuvettes,  N+1 cuvettes
<li> Steinberg's, at 4 C.
* Steinberg's, at 4 C.
<li> Square pulse electroporator (we use the BTX 830 Squarporator)
* Square pulse electroporator (we use the BTX 830 Squarporator)


1)  Put cuvettes on ice, and cool down Steinberg's on ice


2) Trypsinize cells as normal, stop reaction with high serum media and spin down at 1000 rpm, 3 minutes
''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)
|}


3)  Wash pellet with Steinberg's solution and resuspend in 300 µl of Steinberg's, keep on ice


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

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

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