Other important parameters for optimizing electroporation.
Influence of the DNA/RNA
The transfection efficiency of electroporation can be affected by the
concentration, the purity and the size of the molecules used.
a) Influence of nucleic acid concentration:
With the optimal electroporation parameters (osmolarity, voltage, pulse
length), the quality of the results obtained with plasmid concentrations
between 5 g/ml and 20 g/ml is usually satisfactory. The
efficiency of the transfection may be raised by increasing the nucleic
acid concentration, but only within a limited concentration range. Tests
with various different cell lines have shown that only in very few cases
(e.g. when large plasmids were used) do plasmid concentrations in excess
of 20 g/ml lead to an increase in the transfection rate (see Fig.
Figure 1. Transient transfection efficiency in NIH-3T3
cells in relation to the DNA concentration (g/ml). The cells
were electroporated with different concentrations of the pEGFP-N1
plasmid. The transfection rate (max. = 100%) was determined by FACS
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. Therefore, it is recommended
to dissolve the nucleic acid in distilled water before transfection. Any
losses resulting from DNA buffer exchange are usually more than compensated
by the increased transfection efficiency. Irrespective of the preparation
method used, the DNA/RNA should be ultra-pure (A260 nm/A280 nm between 1.7
and 1.9). It is also important to use endotoxin-free DNA for sensitive cells.
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.
Source:Page: All 1 2 Related biology technology :1
. Optimizing Transfection Conditions for Studying Signal Transduction Pathways2
. Optimizing pfuturbo DNA Polymerase Amplification Reactions with
Perfect Match PCR Enhancer3
. Optimizing electroporation parameters4
. Optimizing Büchi® Rotary Evaporator Applications5
. Optimizing electroporation parameters6
. Optimizing Büchi® Rotary Evaporator Applications7
. Optimizing Bchi Rotary Evaporator Applications8
. Optimizing electroporation parameters9
. Optimizing electroporation parameters10
. Optimizing electroporation parameters11
. Optimizing electroporation parameters