Plate cells approx. 24 hours before transfection making sure cells are at optimal concentration (70–90 % confluency).
Allow X-tremeGENE™ 9 DNA Transfection Reagent, DNA and diluent (Opti:MEMR® I Reduced Serum Medium or serum-free medium) to warm to +15° C to +25° C, and vortex gently.
Store X-tremeGENE™ 9 DNA Transfection Reagent at +2 to +8 °C and X-tremeGENE™ HP DNA Transfection Reagent at
-15 to -25 °C.
Bring the vial containing X-tremeGENE™ DNA Transfection Reagent to +15 to +25 °C, and vortex for one second, before removing the desired amount.
Do not aliquot X-tremeGENE™ DNA Transfection Reagent; store remaining transfection reagent in the original glass vials.
Minimize contact of undiluted X-tremeGENE™ DNA Transfection Reagent with plastic surfaces.
After removing the amount required, tightly close the lid of the vial immediately after use.
The minimum amount of X-tremeGENE™ DNA Transfection Reagent: DNA complex for use in a transfection is 100 μL. Complex formation at lower volumes significantly decreases transfection efficiency.
Do not use tubes or microplates made of polystyrene for X-tremeGENE™ DNA Transfection Reagent: DNA complex preparation. When not able to avoid polystyrene materials, make certain to pipet the transfection reagent directly into the serum-free medium, or a reduced-serum medium (that does not contain any serum).
Do not use siliconized pipette tips or tubes.
Make certain that cells are still actively growing at the time of transfection.
Include appropriate controls when performing transfections, by including wells with:
(1) untransfected cells
(2) cells with transfection reagent alone
(3) cells with DNA alone
The optimal ratio of transfection reagent: DNA, and the optimal total amount of complex, can vary according to cell line, cell density, status of cell growth for the assay, and gene expressed.