Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 55)

5.2 DNA
While the majority of electroporation applications involve delivery of plasmid DNA to cells, nearly any type
of molecule can be introduced into cells by electroporation, including RNA, proteins, carbohydrates, and
small molecules, such as nucleoside triphosphates and fluorescent dyes. With few exceptions, when
delivering autonomously replicating plasmids, the highest transformation efficiencies are obtained when
electroporating supercoiled plasmid; this is the case for microorganisms, plant, and animal cells.
However, integration of electroporated plasmid into the host genome is usually most efficient using linearized
plasmid, such as when isolating stable transformants of mammalian cells (Barsoum, 1995), or studying
homologous recombination in Candida (Thompson, et al., 1998), Pichia (Cregg & Russell, 1998), and
Tetrahymena (Gaertig & Gorovsky, 1995). Addition of carrier, such as salmon sperm DNA or plasmid has
also been shown to increase gene expression in some cell lines (Chu, et al., 1987; Showe, et al., 1990).
Although transformation of most cell types has been accomplished using plasmid DNA isolated by a variety
of methods, the plasmid purity has an effect on transformation efficiency. Significantly lower transformation
efficiencies are generated with unpurified miniprep plasmid DNA than with plasmid DNA that has been puri-
fied. Plasmid produced using the Bio-Rad Aurum matrix is as efficient as CsCl-purified plasmid for
transformation of both microorganisms and mammalian cells. However, as long as plasmids used for
are all prepared in the same manner, changes in expression levels are due to differences in transcription or
translation of the gene of interest.
Finally, when electroporating ligation mixtures into E. coli, components in the reactions have been shown
to interfere with transformation (Dower, 1990). Heating the ligation mixture (65°C for 15 min) followed
either by dilution with water (Willson and Gough, 1988), dialysis (Heery and Dunican, 1989; Jacobs et al.,
1990), or ethanol precipitation (Böttger, 1988; Zabarovsky and Winberg, 1990) significantly increases the
transformation efficiency of the DNA.
5.3 Electroporation Media
Many types of microorganisms are most efficiently electroporated in high resistance media
(resistance >3000 ohms). For this reason, when preparing electrocompetent cells, it is impor-
tant to wash cells thoroughly to remove all traces of ions present in the growth media. Failure
to thoroughly remove the growth media from the cells may result in the sample arcing during
electroporation at high field strengths. Cells should be washed at least three times with water
or with non-ionic solutions, such as glucose, glycerol, sucrose, or sorbitol. For many microorganisms,
10–15% glycerol is a convenient electroporation medium, since it is recommended as a cry-
oprotectant for storage of cell cultures.
In some bacterial species, including low (~1 mM) amounts of MgCl
2
in the electrophoresis buffer increases
the transformation efficiency. At least in some bacteria, Mg ++ ions probably maintain the structural integrity
of the cell membrane. For example, Pseudomonas aeruginosa is very sensitive to divalent cation chelators;
incubation of cells in the presence of EDTA destabilizes the outer membrane of the cells (Haque & Russell,
1974). In the absence of Mg
++ ions, the membrane may not be able to re-form following electroporation.
Figs 5.1A and B show the effect of concentration of several biologically important ionic solutions on
sample resistance. Note that: (1) the volume of liquid in the cuvette has a significant effect on sample
resistance—for ionic solutions, sample resistance is inversely proportional to the volume of solution in
the cuvette; (2) the resistance of a solution containing divalent ions is lower than a solution containing
the same concentration of monovalent ions; and (3) the resistance of a buffered solution is affected by
its pH.
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