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X-tremeGENE HP DNA Transfection Reagent Protocol

Cell preparation for transfection

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

Steps prior to transfection

Allow X-tremeGENE 9 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.

  • Place diluent in a sterile tube.
  • Add plasmid DNA. Pipet gently to mix.
  • Add X-tremeGENE HP DNA Transfection Reagent to the diluted DNA.
  • Incubate for 15 min at +15° C to +25° C.
  • Add transfection complex to the cells in a dropwise manner.
  • Gently shake or swirl the wells or flasks to ensure even distribution over the entire plate.
  • Incubate cells for 18 – 72 hours before measuring protein expression.

Volumes of X-tremeGENE 9 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
1 μl
1 μg
5 μl
5 μg
2 : 1 X-tremeGENE HP DNA Transfection Reagent
2 μl
1 μg
10 μl
5 μg
3 : 1 X-tremeGENE HP DNA Transfection Reagent
3 μl
1 μg
15 μl
5 μg


Tips for successful transfections

  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 X-tremeGENE DNA 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.


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