We are having some success in our HEK transfection but want to optimize the protocol to see if we can do better. What steps would you recommend and which do you think would have the most impact?

Answer

Unfortunately, this is a very difficult question to answer, as every step and each procedure plays a role in the success of your transfection. Always start be ensuring that your cells are as healthy as possible and have been cultured under controlled conditions, not letting the cells density get too high, or be too low, at the time of sub-culturing. Cell viability should be very high at the time of transfection. When generating transfection complexes, it is important to use a transfection reagent that has been shown to be compatible with your cells and to lead to high transfection efficiencies. Experiments should be performed to optimize the complexation media used as well as the amount of transfection reagent and plasmid DNA, the temperature of the reaction and the amount of time that complexes are allowed to form before addition to your cells. Also, be sure that highly charged inhibitory components (such as anti-clump) are not present in your transfection media. Thus, transient transfection should be viewed as more of a system in order to obtain optimal results rather than a collection of individual steps.