Eppendorf Multiporator - Electroporation - Manual (Page 12)

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b) Influence of nucleic acid purity
Empirical studies have shown that EDTA and buffer salts such as HEPES or TRIS can drastically reduce transfection
efficiency. We therefore recommend dissolving the nucleic acid in distilled water before transfection. Any losses
resulting from DNA buffer exchange are usually more than compensated for by the increased transfection efficiency.
Irrespective of the preparation method used, the DNA/RNA should be ultra-pure (A
260
nm/A
280
nm ≥ 1.8 ).
It is also important to use endotoxin-free DNA. Otherwise an increase in the DNA concentration will lead to an
increase in the endotoxin content in the cell suspension.
c) Influence of the size of the plasmid
Transfection efficiency is also affected by the size of the individual molecule that is introduced into the cell. This
means that the optimal electroporation parameters that were determined for a certain plasmid, for example, have to
be changed when a larger or smaller plasmid is used.
The temperature has a direct effect on the permeation voltage of the cell membrane as well as on the regeneration of the
membrane following electroporation.
a) Influence of the temperature on the permeation voltage of the cell membrane
Since the permeation voltage at 4 °C is twice that at room temperature, it is essential to take the temperature into
account when determining the optimal field strength of the pulse. Therefore, during electroporation at 4 °C, the
necessary field strength of the pulse is also nearly twice as high as those values for room temperature. However,
mammalian cells are usually electroporated at room temperature.
b) Influence of the temperature on the regeneration of the cell membrane
Incubating cells following electroporation at low temperatures (e.g. 4 °C) slows down the healing process of the cell
membrane. In the case of eukaryotic cells, the resealing of the membrane pores can take half an hour and longer
under these conditions. With certain cell types, this can lead to an increase in the amount of transfection material
absorbed. However, some cells are extremely sensitive to low temperatures, particularly when permeated, and can
suffer from irreversible damage after short incubation times in a cold environment.
In those cases where electroporation at 4 °C leads to higher transfection rates, the cells' chances of survival can be
boosted if they are resuspended in electroporation buffer at 37 °C or room temperature, cooled down to 4 °C and
then transferred into precooled cuvettes. Following electroporation, the cells are incubated at 4 °C for a maximum of
two minutes and then heated to 37 °C.
Electroporation at higher temperatures (e.g. >25 °C) causes the permeated membrane areas to seal up more rapidly,
which accelerates membrane regeneration and thus increases the cell survival rate. However, the transfection rate
may be lower than that obtained when electroporation is carried out at low temperatures.
c) Influence of the temperature on the conductivity of the medium
The temperature has a profound effect on the conductivity of the electroporation buffer. Increasing the temperature
causes the conductivity of a solution to increase as well, which may lead to lower transfection rates. For this reason,
it is advisable not to work at temperatures in excess of 33 °C.
3.6 Influence of the temperature
3 Optimizing the parameters
3 Optimizing the parameters
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