T8198-03 Transfection Reagent (in vitro) is a sterile solution of a cationic polymer in water. The polymer forms compact, stable, positively charged complexes with DNA. These complexes protect DNA from degradation and facilitate gene delivery into eukaryotic cells. T8198-03 Transfection Reagent (in vitro) is ideal for transfection of a variety of cells, including primary and difficult-to-transfect cells. Transfection can be performed in the presence or absence of serum. T8198-03 Transfection Reagent (in vitro) demonstrates superior transfection efficiency and minimal toxicity when compared to lipid- based or other polymer-based transfection reagents. T8198-03 Transfection Reagent (in vitro) for 300-500 in vitro transfections. , Reagents Required but Not Supplied:, Serum-free DMEM, RPMI or other growth medium. , The presence of antibiotics in the medium has no effect on transfection efficiency. General Considerations DNA Quality Requirements:, DNA quality is critical for successful transfection. An A260/A280 ratio of 1.8 or higher is recommended. Endotoxin-contaminated DNA may result in inefficient transfection and cause unacceptably high cellular toxicity. Cell Density: The recommended confluency for adherent cells at the day of transfection is 70-90%. Suspension cells should be plated at an optimal density ensuring their logarithmic growth at the time of transfection. Incubation Time: Transient transgene expression takes place within 2-72 hours after transfection. The optimal incubation time depends on the cell type, promoter strength and expression product, and must be determined experimentally. Choice of Promoter: High transfection efficiency depends both on the transgene promoter and on the cell line used. 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. Transfection Reagent/DNA Ratio: The volume of transfection reagent used depends on the amount of DNA, transgene and cells to be transfected. The ratios presented in the protocols below are starting amounts and can be further optimized for best results. Transfection in the Presence of Serum: The transfection efficiency with T8198-03 Transfection Reagent (in vitro) is equally high in the presence of serum. This is not the case with many other transfection reagents. Antibiotics: Antibiotics do not interfere with both DNA/TurboFect complex formation and cell transfection. Quality Control: Transfection efficiency was tested on HeLa cells using 1ug of eGFP expressing plasmid and 2ul of TurboFect per 5x10e4 cells in 24-well plate. Transfection efficiency, i.e. the percentage of transfected cells, is 90±10% as estimated by Flow Cytometry. Storage and Stability: May be stored at 4°C. For long-term storage, aliquot and store at 4°C. Do not freeze. Aliquots are stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial prior to removing the cap. Further dilutions can be made in assay buffer.