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Optimizing electroporation parameters

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. 1.).

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 analysis. 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.



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