I. DNA Quality Requirements
DNA quality is critical for successful transfection. Endotoxin-contaminated DNA may result in inefficient transfection and cause unacceptably high cellular toxicity. For DNA an A260/A280 ratio of 1.8, or greater is recommended.
II. Cell Density
The recommended confluency for adherent cells on the day of transfection is 70-90% for TurboFect reagents.
III. Incubation Time
Transient transgene expression takes place within 2-72hours after DNA transfection. The optimal time depends on the cell type, promoter strength and expression product, and has to be determined experimentally. The recommended incubation time of cells with TurboFect/protein complexes is 2 hours.
IV. Choice of Promoter
High transfection efficiency depends both on the transgene promoter and on the cell line used. The cytomegalovirus (CMV) promoter is commonly used for high gene expression in a variety of cell lines. Other promoters, such as those from simian virus (SV40) and from Rous sarcoma virus (RSV) can also be used.
V. Transfection Reagent/Biomolecule Ratio
The amount of transfection reagent used in transfection depends on the amount of DNA, siRNA or protein and cells to be transfected. The ratios presented in the protocols are starting ratios and can be further optimized for the best results.
VI. Transfection in the Presence of Serum
Nucleic acid transfection efficiency using Fermentas transfection reagents is consistently high in the presence of serum. The presence of serum may reduce protein transfection efficiency by up to 50%. Therefore, protein transfection in serum-free medium is recommended for best results.
Gentle centrifugation of tissue culture plates for 5minutes at 280 x g after addition of the polyplexes can improve transfection efficiency.