( ficiency is lower than expected ...)Transfection Reagent Selector Kit Handbook

ficiency is lower than expected and cytotoxicity acceptably low, increase the overall amount of Transfection ReagentDNA complex. See the example pipetting scheme. Incubation time for gene expression is sub-optimal Different cell types achieve maximal expression levels at different times posttransfection. This should be kept in mind when determining the 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. Vector influence 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. Cell density at the time of Transfection ReagentDNA complex addition is too high If cell density is too high during 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 4080%. Poor DNA quality 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 QIAGEN or QIAfilter Plasmid Kits or an equivalent method. For endotoxinsensitive cell lines
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