Critical parameters for successful transfection
Quantity of nucleic acids
Amount of transfection reagent
Transfection complex formation and complex incubation time
Many chemical transfection reagents have an ideal time window, in which a transfection complex of optimal diameter is formed. This is typically between 5 and 30 minutes, depending upon the nature of the reagent. Refer to the reagent manufacturer’s recommendations.
In general, transfection reagents need to be in contact with cells for a period of time before additional medium is added or the medium is replaced (to help minimize toxic effects of the reagent). The optimal transfection time depends on the cell line, transfection reagent, and nucleic acid used.
In some instances, plating cells onto wells or plates containing transfection complexes may result in increased transfection efficiency, compared to the traditional approach of adding transfection complexes to an established culture. An additional benefit to such reverse transfection protocols is that seeding and transfecting cells on the same day shortens the experimental timeline by a full day.