Oligofectamine™ Reagent is a proprietary formulation for the transfection of oligonucleotides 1 and short interfering RNA (siRNA) 2,3 into eukaryotic cells. The reagent formulation has been changed to enhance its stability at cold temperatures (+4°C) while continuing to provide the highest specific activity and lowest non-specific effects on cell growth.
Performance may be enhanced in some assays.
Guidelines for Transfection
- Use the procedure at www.lifetechnologies.com/rnai; click on Protocols.
- Use an initial oligonucleotide concentration of 200 nM for transfection. Optimal oligonucleotide concentrations may range from 50-250 nM; optimize as necessary. Always include a control oligonucleotide in the experiment to assess non-specific effects.
- Prepare complexes using the amount of oligonucleotide and Oligofectamine™ recommended on below Optimization may be necessary. Note: We recommend using Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute Oligofectamine™ and oligonucleotide before complexing.
- Transfect cells at 30-50% confluence. Optimize as necessary. Maintain the same seeding conditions between experiments. Use cells within 20 passages of optimization.
- Do not add antibiotics to media during transfection as this causes cell death.
- For optimal results, perform transfection in medium without serum. Transfection in the presence of serum may be tested, if desired. Test any serum-free medium for compatibility with Oligofectamine™.
- One day before transfection, plate cells in 100 µl of growth medium without antibiotics so that cells will be 30-50% confluent at the time of transfection.
- For each transfection sample, prepare complexes as follows:
- Dilute 1 µl of a 20 µM stock oligonucleotide in 16 µl of Opti-MEM® I Reduced Serum Medium (or other medium) without serum. Mix gently.
- Mix Oligofectamine™ gently before use, then dilute 0.4-0.8 µl in Opti-MEM® I Medium (or other medium) without serum to a final volume of 3 µl. Mix gently and incubate for 5-10 minutes at room temperature.
- Combine the diluted oligonucleotide with diluted Oligofectamine™ (total volume = 20 µl). Mix gently and incubate for 15-20 minutes at room temperature (solution may appear cloudy).
Scaling Up Transfections
Relative surf. area vs. 96-well
Oligo (µl of 20 µM stock) & dilution vol. (µl)
Oligofectamine™ (µl) & final dilution vol. (µl)
Plating medium vol.
Total vol. per well
Added vol. medium with 3X serum
1 µl in 16 µl
0.4-0.8 µl to 3 µl
2.5 µl in 40 µl
1-2 µl to 7.5 µl
5 µl in 85 µl
1-3 µl to 10 µl
10 µl in 175 µl
2-4 µl to 15 µl
2. Elbashir, S.M., et al. (2001) Nature 411, 494.
3. Harborth, J., et al. (2001) J. Cell Sci. 114, 4557.