Protocols for Transfecting the Most Common Cell Lines with X-tremeGENE™ Transfection Reagent

Transfection of CHO-K1 Cells

Plate CHO-K1 cells at a density of 1.5 X 104 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (65 – 75% confluency). Incubate cell cultures overnight.

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 200 μl diluent in a sterile tube.
  • Add 6 μl X-tremeGENE 9 DNA Transfection Reagent to the diluent. Pipet gently to mix.
  • Add 2 μg plasmid DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 5 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: CHO-K1 cells were successfully transfected with pcDNA3.1-GFP plasmid using using X-tremeGENE 9 DNA Transfection Reagent.

Transfection of COS-7 Cells

Plate COS-7 cells at a density of 8.0 – 9.0 X 103 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (75 – 85% confluency). Incubate cell cultures overnight.

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.

  • Place 200 μl diluent in a sterile tube.
  • Add 6 μl X-tremeGENE 9 DNA Transfection Reagent to the diluent. Pipet gently to mix.
  • Add 2 μg plasmid DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 5 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: COS-7 cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

Transfection of HeLa Cells

Plate HeLa cells at a density of 1.5 X 104 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (70 – 90% confluency). Incubate cell cultures overnight.

Allow X-tremeGENE 9 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 200 μl diluent in a sterile tube.
  • Add 6 μl X-tremeGENE 9 DNA Transfection Reagent to the diluent. Pipet gently to mix.
  • Add 2 μg plasmid DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 5 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: HeLa cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

Transfection of NIH-3T3 Cells

Plate NIH-3T3 cells at a density of 1.5 X 104 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (70 – 80% confluency). Incubate cell cultures overnight.

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 200 μl diluent in a sterile tube.
  • Add 6 μl X-tremeGENE 9 DNA Transfection Reagent to the diluent. Pipet gently to mix.
  • Add 2 μg plasmid DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 5 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: NIH-3T3 cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

Transfection of HEK-293 Cells

Plate HEK-293 cells at a density of 3.0 – 3.5 X 104 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (50 – 70% confluency). Incubate cell cultures overnight.

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.

  • Place 200 μl diluent in a sterile tube.
  • Add 2 μg plasmid DNA. Pipet gently to mix.
  • Add 6 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 10 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: HEK-293 cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE HP DNA Transfection Reagent.

Transfection of PC-3 Cells

Plate PC-3 cells at a density of 1.0 – 1.2 X 104 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (75 – 85% confluency). Incubate cell cultures overnight.

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.

  • Place 200 μl diluent in a sterile tube.
  • Add 2 μg plasmid DNA. Pipet gently to mix.

Add 2 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (1:1 ratio of reagent to DNA). Pipet gently to mix.

Incubate for 15 – 30 min at +15° C to +25° C.

  • Add 10 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: PC-3 cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE HP DNA Transfection Reagent.

Transfection of HCT 116 Cells

Plate HCT 116 cells at a density of 3.0 – 4.0 X 105 cells/well. Plate cells in a volume of 1 ml complete growth medium per well in a 12-well plate 18 – 24 hours before transfection (80% confluency). Incubate cell cultures overnight.

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.

  • Place 100 μl diluent in a sterile tube.
  • Add 1 μg plasmid DNA. Pipet gently to mix.
  • Add 2 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (2:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 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 24 – 72 hours before measuring protein expression.

Transfection of A549 Cells

Plate A549 cells at a density of 2.8 X 104 cells/well. Plate cells in a volume of 150 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (70% confluency). Incubate cell cultures overnight.

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.

Place 500 μl diluent in a sterile tube.
Add 5 μg plasmid DNA. Pipet gently to mix.

  • Add 10 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (2:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 15 μl 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 24 – 72 hours before measuring protein expression.

Transfection of MCF-7 Cells

Plate MCF-7 cells at a density of 1.2 – 1.8 X 104 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (70 – 90% confluency). Incubate cell cultures overnight.

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 200 μl diluent in a sterile tube.
  • Add 6 μl X-tremeGENE 9 DNA Transfection Reagent to the diluent. Pipet gently to mix.
  • Add 2 μg plasmid DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 10 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: MCF-7 cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

Transfection of HuH-7 Cells

Plate HuH-7 cells at a density of 1.0 X 105 cells/well. Plate cells in a volume of 2.5 ml complete growth medium per well in a 6-well plate 18 – 24 hours before transfection. Incubate cell cultures overnight.

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.

  • Place 100 μl diluent in a sterile tube.
  • Add 1.5 μg plasmid DNA. Pipet gently to mix.
  • Add 1.5 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (1:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • One hour before adding the complexes to cells, refresh the medium with 1.5 ml of Opti-MEM I Reduced Serum Medium.
  • Add 100 μl 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.
  • The next day refresh the medium with complete growth medium.
  • Incubate cells for 24 – 72 hours before measuring protein expression.

Transfection of U-2 OS Cells

Plate U-2 OS cells at a density of 2.5 – 3.0 X 104 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (70 – 90% confluency). Incubate cell cultures overnight.

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.

  • Place 200 μl diluent in a sterile tube.
  • Add 2 μg plasmid DNA. Pipet gently to mix.
  • Add 4 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (2:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 10 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: U-2 OS cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE HP DNA Transfection Reagent.

Transfection of HepG2 Cells

Plate HepG2 cells at a density of 2.0 – 2.5 X 104 cells/well. Plate cells in a volume of 100 μl complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (60 – 70% confluency). Incubate cell cultures overnight.

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 500 μl diluent in a sterile tube.
  • Add 30 μl X-tremeGENE 9 DNA Transfection Reagent to the diluent. Pipet gently to mix.
  • Add 10 μg plasmid DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 5 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: HepG2 cells were successfully transfected with pcDNA3.1-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

Transfection of Neuro-2a Cells

Protocol provided by a customer.
Plate Neuro-2a cells at a density of 1.0 X 105 cells/well. Plate cells in a volume of 500 μl complete growth medium per well in a 24-well plate 24 hours before transfection (30 – 40% confluency). Incubate cell cultures overnight.

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 100 μl diluent in a sterile tube.
  • Add 8 μl X-tremeGENE 9 DNA Transfection Reagent to the diluent. Pipet gently to mix.
  • Add 2 μg plasmid DNA. (4:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 100 μl 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 24 – 72 hours before measuring protein expression.

Note: Data and experimental conditions included in “Protocols provided by customers” are the sole responsibility of the customers that have submitted them. Roche was neither involved in establishing the experimental conditions nor in defi ning the criteria for the performance of the specifi c assays. Roche therefore cannot take any responsibility for performance or interpretation of results obtained for the biological target parameter(s) described by the authors or other users using a similar experimental approach.expression.

Experimental result: Neuro-2a cells were successfully transfected with CMV6-GFP plasmid using X-tremeGENE 9 DNA Transfection Reagent.

Transfection of Primary MEF Cells

Plate primary MEF cells at a density of 0.5 – 1.0 X 105 cells/well. Plate cells in a volume of 1 ml complete growth medium per well in a 12-well plate 18 – 24 hours before transfection (60% confluency). Incubate cell cultures overnight.

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.

  • Place 100 μl diluent in a sterile tube.
  • Add 1 μg plasmid DNA. Pipet gently to mix.
  • Add 4 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (4:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 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 24 – 72 hours before measuring protein expression.

Transfection of Human Primary Fibroblasts from Pre-Skin

Plate human primary fibroblast cells at a density of 1.0 X 105 cells/well. Plate cells in a volume of 2 ml complete growth medium per well in a 6-well plate 18 – 24 hours before transfection (40 – 50% confl uency). Incubate cell cultures overnight.

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.

  • Place 200 μl diluent in a sterile tube.
  • Add 2 μg plasmid DNA. Pipet gently to mix.
  • Add 6 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 200 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: Human primary fibroblasts were successfully transfected with GFP-encoding plasmid using X-tremeGENE HP DNA Transfection Reagent.

Murine Mesenchymal Stem Cells EK8

Plate EK8 cells at a density of 3.0 X 104 cells/well. Plate cells (3 X 105 cells/ml) in a volume of 100 μl complete growth medium per well in a 96-well plate 24 hours before transfection (50% confluency). Incubate cell cultures overnight.

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.

  • Place 392 μl diluent in a sterile tube.
  • Add 8 μg plasmid DNA. Pipet gently to mix.
  • Add 8 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (1:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 10 μl 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 24 – 72 hours before measuring protein expression.

Transfection of Human Mesenchymal Stem Cells

Plate hMSCs at a density of 8.0 X 104 cells/well. Plate cells in a volume of 2 ml complete growth medium per well in a 6-well plate 18 – 24 hours before transfection (50% confluency). Incubate cell cultures overnight.

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.

  • Place 200 μl diluent in a sterile tube.
  • Add 2 μg plasmid DNA. Pipet gently to mix.
  • Add 6 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 200 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: human mesenchymal stem cells were successfully transfected with GFP-encoding plasmid using X-tremeGENE HP DNA Transfection Reagent.

Transfection of HEK-293T Cells for Lentiviral Production

Plate HEK-293T cells at a density of 5.0 X 105 cells/well. Plate cells in a volume of 2 ml complete growth medium per well in a 6-well plate 18 – 24 hours before transfection (60 – 80% confluency). Incubate cell cultures overnight.

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.

  • Place 180 μl diluent in a sterile tube.
  • Add 2 μg plasmid DNA. (20 μl of a plasmid DNA mixture in a molar ratio of 1: 2: 2 = library plasmid : packaging plasmid : envelope plasmid). Pipet gently to mix.
  • Add 6 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (3:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 min at +15° C to +25° C.
  • Add 200 μl 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 24 – 72 hours before measuring protein expression.

Experimental result: HEK-293T cells were successfully transfected with pGIPZ-eGFP plasmid using X-tremeGENE HP DNA Transfection Reagent.

Transfection of SF9 Insect Cells

Plate SF9 cells at a density of 0.5 X 106 cells/well. Plate cells in a volume of 1 ml complete growth medium per well in a 12-well plate immediately before transfection. Incubate cell cultures overnight.

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.

  • Place 100 μl diluent in a sterile tube.
  • Add 1 μg plasmid DNA. Pipet gently to mix.
  • Add 8 μl X-tremeGENE HP DNA Transfection Reagent to the diluted DNA. (8:1 ratio of reagent to DNA). Pipet gently to mix.
  • Incubate for 15 – 30 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 24 – 72 hours before measuring protein expression.

For life science research only. Not for use in diagnostic procedures.

Please be aware that the protocols are suggestions and that optimal conditions must be determined empirically.

  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.

Materials

     
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