The following paramenters can be optimized to increase transfection efficiency when using Effectene Transfection Reagent:
Optimize the Effectene Reagent to DNA ratio
If the ratio of Effectene Reagent to DNA is suboptimal, the overall charge of the complexes may be negative, neutral or strongly positive, which can lead to inefficient adsorption to the cell surface. Optimize the Effectene Reagent to DNA ratio according to the section " Transfection Optimization" of the Effectene Transfection Reagent Handbook.
Increase the amount of Effectene-DNA complex
If the transfection efficiency is lower than expected and cytotoxicity acceptably low, increase the overall amount of Effectene-DNA complexes. See the pipetting scheme in the section "Transfection Optimization" of the Effectene Transfection Reagent Handbook for details.
Optimize the incubation time for gene expression
Different cell types achieve maximal expression levels at different times post-transfection. This should be kept in mind when determining length of incubation after transfection. If the time point of maximal expression is not known for a particular cell line, a time course experiment may be necessary.
Consider vector influences
Factors such as the promoter, origin of replication, and plasmid size influence gene expression rate. The optimal quantity of plasmid DNA used for transfection is dependent on the expression rate of the plasmid.
Optimize cell density at the time of Effectene-DNA complex addition
If cell density is too high at the time of transfection-complex addition, cells may not be at the optimal phase of growth. This can lead to insufficient uptake of the complexes into the cells or insufficient expression of the gene of interest. For adherent cells, the optimal confluency at the time of transfection complex addition is normally 40-80%.
Use high-quality DNA Plasmid DNA used for transfection should be of high quality
Impurities present in the DNA preparation can potentially lower transfection efficiency. DNA should be purified using HiSpeed, QIAfilter, or QIAGEN Plasmid Kits or an equivalent method. For endotoxin-sensitive cell lines and primary cells, we recommend using DNA purified with EndoFree Plasmid Kits to ensure the highest transfection efficiencies.