Transfection tips

The choice of transfection reagent depends on both the molecule you wish to transfect (see table below) and your target cells. Some cell types (e.g., HEK293) are easy to transfect with almost any reagent, while others (e.g., lymphocytes and neurons) are notoriously difficult to transfect. According to ongoing customer feedback surveys, Xfect transfection reagents perform exceptionally well with a broad range of cells (for example, see our list of cell lines successfully transfected with the Xfect plasmid-transfection reagent).

Use the following table to select the best reagent for your application. Refer to each reagent's user manual for recommended storage conditions and information about sensitivity to serum/antibiotics in the culture media.

Notes:

• If you are transfecting neural or neuronal cells, we recommend using the CalPhos Mammalian Transfection Kit.
• We also offer adenoviral, lentiviral, and retroviral gene delivery systems.

The overall health of target cells at the time of transfection is a key factor for transfection efficiency. We recommend using a 60–75% confluent culture for plasmid transfection—this is when cells are optimally active, and the risk of reagent toxicity is low. Xfect products are highly efficient with little to no cytotoxicity, further increasing your chances of success.

Cell culture quality decreases with repeated passages, so keep the cell passage number low. Passage newly thawed cells at least twice and plate prior to transfection as instructed in the reagent's user manual.

Using excess or insufficient amounts of cargo and reagent can have adverse effects on transfection efficiency. Refer to your product manual for the optimal cargo:reagent ratio and maintain the recommended ratio across any experimental scale and/or culture surface area.

Use only high-purity DNA (OD_260/280 >1.8) for plasmid transfection. We recommend Macherey-Nagel NucleoBond Xtra Midiprep and Maxiprep kits—including the endotoxin-free kits—for transfection-grade plasmid purification (see the tech note).

The optimal incubation period for cargo and reagent varies with each transfection experiment and must be determined empirically. Since prolonged exposure to transfection reagents may result in cell toxicity, it is critical to stay within the time range recommendations advised in your manual.

• Reporter assays: you can use a variety of methods to measure transfection efficiency, including fluorescent and non-fluorescent reporter assays, qRT-PCR, and Western blotting. Please note that some assays are more rapid and sensitive than others.
• When to run your assay: most reporter assays are performed 1–3 days after transfection. The exact timing will depend on the type of cells you are transfecting, the reporter you are using, and other factors specific to your transfection experiment. You must determine the best time to run your assay empirically.

There will always be some variability in transfection efficiencies, but optimizing transfection parameters for each cell type is crucial to minimizing this variability. Once you optimize the transfection parameters, they should be kept consistent in order to obtain reproducible results from one experiment to the next.