Bio-Rad MicroPulser™ Electroporator - Manual (Page 10)

at 5 milliseconds when working with high-resistance samples. For these organisms, the
MicroPulser has pre-programmed settings for delivery of the correct voltage when
electroporating E. coli in either 0.1 or 0.2 cm cuvettes, or when electroporating S. cerevisiae
in either 0.2 or 0.4 cm cuvettes.
3.1 Cell Growth
For most bacterial species, the highest transformation efficiencies are obtained when cells
are harvested in early to mid-log growth. For E.coli, as the cells reach stationary phase, the
transformation efficiency will decline precipitously (Dower, 1990). In contrast, most yeast
species are generally harvested in mid- to late-log growth. For S. cerevisiae, the
transformation efficiency increases as much as 60-fold from early to late-log cultures (Becker
and Guarente, 1991). The optimal portion of the growth phase to harvest cells is generally
dependent on the cell type. When preparing competent cells of a new species it is generally
best to employ conditions worked out for use with the same genus. Suggestions for factors to
consider and general methods for producing electrocompetent cells are discussed in the
articles by Dower et al. (1992) and Trevors et al. (1992).
3.2 DNA
While the majority of electroporation applications involve delivery of plasmid DNA to
cells, it should be mentioned that nearly any type of molecule can be introduced into cells by
electroporation, including RNA, proteins, carbohydrates, and small molecules. With few
exceptions, when delivering autonomously replicating plasmids, the highest transformation
efficiencies are obtained when electroporating supercoiled plasmid. However, electroporating
plasmid that will integrate into the host genome is usually most efficient using linear
plasmid. For example, Candida, Pichia, and Tetrahymena are transformed more efficently
when transformed with linearized than with supercoiled integrating plasmids.
In both E. coli and Listeria monocytogenes, the transformation efficiency of relaxed
circular plasmid is only slightly lower than that of supercoiled plasmid (Leonardo and Sedivy,
1990, Park and Stewart, 1990). However, linear plasmid is about 10
3 - 10 4 -fold less efficient
than the corresponding circular plasmid in both E. coli and Streptococcus pyogenes
(Shigekawa and Dower, 1988, Simon and Ferretti, 1991). Electroporation efficiency per mole
of plasmid generally decreases as the plasmid size increases in numerous species, including
E. coli (Leonardo and Sedivy, 1990, Siguret et al., 1994), Pseudomonas aeruginosa (Dennis
and Sokol, 1995), and Streptococcus thermophilus (Somkuti and Steinberg, 1988). However,
in some species, including Lactococcus lactis (Holo and Nes, 1995), Enterococcus faecalis
(Cruz-Rodz and Gilmore, 1990), and Clostridium perfringens (Allen and Blaschek, 1990),
transformation efficiency appears to be independent of plasmid size up to 20–30 kb.
Although transformation of most microorganisms 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 purified by a variety of procedures. Plasmid
produced using the Bio-Rad Quantum matrix is as efficient as CsCl-purified plasmid for
transformation of microorganisms.
Generally, for all types of microorganisms, the frequency of transformation increases
with inceasing DNA concentration in the electroporation buffer. For E. coli, the frequency of
transformation (transformants/survivor) is dependent on DNA concentration over at least six
orders of magnitude (10 pg/ml to 7.5 µg/ml); within this range the DNA concentration
determines the probablility that a cell will be transformed. At the higher DNA concentrations,
up to 80% of the survivors are transformed (Dower et al., 1988). Because the number of
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