Bio-Rad Gene Pulser MXcell™ Electroporation System - Manual (Page 51)

Gene Pulser MXcell™ System Manual | Factors Affecting Electroporation
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Time Constant and Number of Pulses
While square waves do not report a time constant, they instead are determined by the pulse
duration (or pulse length), which is a time in milliseconds that is programmed into the
instrument being used. It is possible to use shorter or longer pulse durations when
optimizing square wave electroporation. Generally small increments are used. You may
want to test 5 msec lower and higher than the original pulse duration Additionally, it has
been observed that with square wave electroporation, cells might benefit from multiple,
shorter pulses. For example, if the optimal pulse duration is 20 msec, it may be possible to
further optimize by giving two pulses of 10 msec duration each.
The time constant in exponential waveforms is directly related to the resistance of the
sample and the resistance setting used on the electroporator, as well as the capacitance
setting on the electroporator. Resistance of the sample can be affected by either changing
the sample volume or using an electroporation buffer with a higher or lower ionic strength.
Decreasing the sample volume leads to an approximately proportional increase in sample
resistance, thus nearly doubling the time constant.
Cell Growth
For mammalian cells, the highest expression following electroporation is obtained when
cells are in mid-log phase growth (Anderson et al.,1991). Healthy cells transfect better than
poorly maintained cells. Routinely subculturing cells before they become overcrowded or
unhealthy will minimize experimental variability in continuous cell lines. Since cells may
gradually change in culture, using cells within a defined passage number and adhering to
strict protocols, including parameters for intervals between plating and transfecting cells,
will improve experimental reproducibility. It is also important that the cells be healthy and
not contaminated with mycoplasma.
The highest gene expression following electroporation is obtained using cells which are
actively growing and dividing rather than in stationary growth phase. For optimum cell
recovery, the cell density in each well should be in the range of 10
6 -10 7 cells/ml; higher cell
concentrations may result in undesirable cell fusion.
Nucleic Acid and Biomolecules
The transfection efficiency of electroporation can be affected by the concentration, the
purity, and size of the molecules used.
While the majority of electroporation applications involve delivery of plasmid DNA and
siRNAs 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.
DNA
With few exceptions, when delivering autonomously replicating plasmids, the highest
transformation efficiencies are obtained when electroporating supercoiled plasmid.
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), Addition of a 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).