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
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
- 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.
Materials