X-tremeGENE™ HP DNA Transfection Reagent Protocol

Cell Preparation for Transfection

Plate cells approximately 24 hours before transfection, making sure cells are at optimal concentration (70–90 % confluency).

Steps Before Transfection

1. Allow X-tremeGENE™ HP DNA Transfection Reagent, DNA and diluent (Opti:MEM^®I Reduced Serum Medium or serum-free medium) to warm to +15 °C to +25 °C, and vortex gently.
2. Place diluent in a sterile tube.
3. Add plasmid DNA. Gently pipette up and down to mix.
4. Add X-tremeGENE™ HP DNA Transfection Reagent to the diluted DNA.
   
5. Incubate for 15 minutes at +15 °C to +25 °C.
6. Add transfection complex to the cells in a dropwise manner.
7. Gently shake or swirl the wells or flasks to ensure even distribution over the entire plate.
8. Incubate cells for 18 – 72 hours before measuring protein expression.

Volumes of X-tremeGENE™ HP DNA Transfection Reagent and amounts of DNA for various ratios

	Ratio Transfection Reagent : DNA	Minimal transfection complex volume	Volume for a whole 96-well plate
	Serum-free medium to a final volume of	100 μL	500 μL
1 : 1	X-tremeGENE™ HP DNA Transfection Reagent DNA	1 μL 1 μg	5 μL 5 μg
2 : 1	X-tremeGENE™ HP DNA Transfection Reagent DNA	2 μL 1 μg	10 μL 5 μg
3 : 1	X-tremeGENE™ HP DNA Transfection Reagent DNA	3 μL 1 μg	15 μL 5 μg

Tips for Successful Transfections using the X-tremeGENE™ DNA Transfection Reagent

1. Store X-tremeGENE™ 9 DNA Transfection Reagent at +2 to +8 °C and X-tremeGENE™ HP DNA Transfection Reagent at -15 to -25 °C.
2. Bring the vial containing X-tremeGENE™ DNA Transfection Reagent to +15 to +25 °C, and vortex for one second, before removing the desired amount.
3. Do not aliquot X-tremeGENE™ DNA Transfection Reagent; store remaining transfection reagent in the original glass vials.
4. Minimize contact of undiluted transfection reagent with plastic surfaces.
5. After removing the amount required, tightly close the lid of the vial immediately after use.
6. 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.
7. 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).
8. Do not use siliconized pipette tips or tubes.
9. Make certain that cells are still actively growing at the time of transfection.
10. Include appropriate controls when performing transfections, by including wells with:
    (1) untransfected cells
    (2) cells with transfection reagent alone
    (3) cells with DNA alone
11. 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.