Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 56)

The addition of even small concentrations of ionic compounds significantly reduces the resistance of
high resistance samples and may cause arcing. Residual salt from ethanol precipitation of DNA should
be removed by washing the DNA pellet prior to dissolving it in either water or Tris-EDTA. Table 5.1
shows that adding a solution of plasmid in 10 mM Tris, pH 8.0 –1 mM EDTA to water does reduce the
sample resistance. This should be considered when electroporating bacterial samples in high-resistance
media. DNA may be used directly from enzyme reactions for transformation, but the final salt concentration
in the electroporation sample should be kept below ~5 meq for high-voltage operation.
5.4 Temperature
The temperature at which cells are maintained during electroporation would be expected to have a role in
the efficiency of the electroporation process for several reasons. First, since passing an electric pulse
through the cells results in heating, keeping the cells at a low temperature during the pulse might reduce
the heating and therefore increase the cell viability. Second, since electroporation involves the transient for-
mation 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. Third, changing the temperature of
a solution changes its conductivity. The conductivity of the media increases with increasing temperature,
resulting in a decrease in the media resistance and a decrease in the time constant,
τ. Finally, diffusion
rate is highly dependent on temperature, and 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. Most bacterial cells are most electrocompetent
when maintained at <4°C from the time of harvest until pulsing. The transformation efficiency of E. coli
decreases by ~100-fold if pulsed at 20°C (Shigekawa & Dower, 1988); however, electrocompetent S.
aureus should be incubated at room temperature prior to electroporation (Lee, 1995). For most mammalian
cells, electroporation is most efficient when cells are maintained at room temperature both before and
after the pulse (Chu, et al., 1987), although some cell types are more efficiently transformed at low
temperature (Potter, et al., 1984).
50