Bio-Rad Gene Pulser MXcell™ Electroporation System - Manual (Page 53)

Gene Pulser MXcell™ System Manual | Factors Affecting Electroporation
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Gene Pulser electroporation buffer is universal and can be used with most cell lines
including primary cells. The buffer works well with both siRNA and plasmid DNA, and
contains components that enhance transfection efficiency and maintain overall cell health
and viability.
Gene Pulser Electroporation Buffer is lower in ionic strength than PBS (it has higher
resistance), thus adjustments need to be made when switching from a protocol using a
traditional low resistance buffer. The recommended starting point from which to optimize is
to decrease the specified capacitance by 50%. Alternatively, the resistance setting on the
electroporator may be reduced to 20% of the original protocol, while maintaining all other
parameters constant.
Temperature
Electroporation of some cell types has been reported to be more efficient at 0-4 o C, in other
cases, room temperatures yields better results. Temperature may affect physical properties
of the membrane, and influences the rate of the duration of the permeabilized state.
Electroporated cells may remain permeable for several hours if low temperatures are
maintained. Loss of permeability is retarded when cells are maintained at 0
o C, but occurs
within a matter of minutes when cells electroporated at 0 o C are subsequently incubated at
37 o C.
The temperature at which cells are maintained during electroporation is expected to have a
role in the efficiency of the electroporation process for several reasons:
• Since passing an electric pulse through the cells results in heating, keeping the
cells at a low temperature during the pulse might reduce heating and therefore
increase cell viability
• Electroporation involves the transient formation of pores in the cell membrane,
keeping cells at a low temperature after the pulse might allow the pores to remain
open longer, giving the DNA in the medium more time to enter the cells.
Alternatively, a higher temperature may speed pore closure and increase cell
viability
• Changing the temperature of a solution changes its conductivity. The conductivity
of the medium increases with increasing temperature, resulting in a decrease in
the medium resistance and a decrease in the time constant
• Diffusion rate is highly dependent on temperature therefore keeping cells at a low
temperature would be expected to reduce the diffusion of molecules across the
cell membrane. In practice, the most efficient temperature at which to
electroporate cells must be determined empirically
For most mammalian cells, electroporation is most efficient when cells are maintained at
room temperature before and after the pulse (Chu, et al. 1987), although some cell types are
more efficiently transformed at low temperature (Potter, et al. 1984).
NOTE: When using Gene Pulser electroporation buffer, electroporation should
be performed at room temperature.