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Product FAQ

Does the method of generating lipid-DNA complex affect transfection efficiency?

Answer

YES. Please follow the recommended procedure for each one of the reagents, found in the product manual.

Answer Id: E3127

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Product FAQ

What are the different methods available for transfection?

Answer

A wide variety of gene delivery techniques are available to introduce plasmid DNA, siRNA or duplex RNAi, oligonucleotides, and RNA into eukaryotic cells for a variety of research and drug discovery applications. A review of these techniques with pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).

Answer Id: E8960

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Product FAQ

What types of molecules can be transfected with cationic lipid reagents?

Answer

Lipid reagents can be used to transfect DNA, RNA, oligonucleotides and siRNA. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb. Lipofectin™ and Lipofectamine™ 2000 Reagent have also been used to transfect cells with proteins (Beta galactosidase and T3 polymerase).

Answer Id: E3128

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Product FAQ

What recommendations do you have for selecting a transfection reagent?

Answer

Please refer to the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to make the right choice.

Answer Id: E8961

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Product FAQ

Is it necessary to use serum-free medium during lipid transfection?

Answer

It is not necessary to use serum-free medium during lipid transfection. However, it is critical to form the lipid:nucleic acid complex in the absence of serum, because proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serum-containing medium.

Answer Id: E8997

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Product FAQ

Can antibiotics be used in media during transfection?

Answer

We discourage using any antibiotics during transfection (e.g. Geneticin™, Hygromycin, Gentamycin, Penicillin, etc). There can be higher cell death when antibiotics are present during transfection. Even though some such as penicillin and streptomycin are not toxic to eukaryotic cells in a healthy culture, during transfection the cell permeability increases so that much higher levels of antibiotics get into cells. For stable transfections, wait at least 24-48 h after transfection before adding selective antibiotics.

Answer Id: E3129

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Product FAQ

Are cell density (% confluency) and passage number important considerations for transfection?

Answer

Yes. Cell density will affect transfection performance. Lipofectamine™ 3000, Lipofectamine™ 2000, and Lipofectamine™ LTX/PLUS provide excellent transfection performance at confluencies between 70 and 90%, while some toxicity may be observed at confluencies lower than this. Lipofectamine™ RNAiMAX works best at confluencies between 60 and 80%.

Passage number may affect transfection experiments. We recommend consistent splitting and plating of cells. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20-30 passages. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen. Please refer to the Gibco™ Cell Culture Basics handbook (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics.html) for proper guidelines for culturing and passaging cells.

Answer Id: E8962

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Product FAQ

Can antibiotics be used in the medium during transfection?

Answer

Yes, antibiotics (penicillin-streptomycin) can be used in the medium during transfection. We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For certain cell types that are sensitive to transfection or have toxicity issues, omitting antibiotics may improve results, however. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.

Answer Id: E8998

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Product FAQ

Is it necessary to use serum-free media during lipid transfection?

Answer

Not in all cases. What is essential is to form the lipid:nucleic acid complex in the absence of serum, because proteins can interfere with complex formation. Once the complexes are formed, they can be added to cells in serum containing medium. For optimal results, with Lipofectin™ perform transfection in medium without serum.

Answer Id: E3131

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Product FAQ

Can I use the same amount of any transfection reagent for different cell lines?

Answer

No. The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use. The protocol that is supplied with the product will provide you with an optimal range of transfection reagent to use per well. During product development, this range was determined to work well across a variety of cell lines. If you are still not achieving the performance you desire in your particular cell line, further optimization may be necessary.

Answer Id: E8963

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Product FAQ

How do cationic lipids compare with calcium phosphate in transfection efficiency?

Answer

For many cell types, higher efficiencies are observed with cationic lipids than with calcium phosphate. Also, cationic lipid data are more reproducible from experiment to experiment. Calcium phosphate is inexpensive however, but pH variation as little as 0.2 can reduce transfection efficiency significantly.

Answer Id: E3137

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Product FAQ

What is the stability of your transfection reagents?

Answer

Our transfection reagents are shipped under ambient conditions and should be stored at 4 degrees C immediately upon receipt. We guarantee the performance of the product, if stored and handled properly, for one year from date of receipt unless otherwise stated on the tube label or COA. We do not recommend freezing transfection reagents, as this usually decreases transfection performance. Please see this white paper (http://tools.thermofisher.com/content/sfs/brochures/cms_103226.pdf) on ambient shipping of Lipofectamine™ transfection reagents.

Answer Id: E8988

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Product FAQ

I am getting very low transfection efficiency. Can you please provide some troubleshooting tips?

Answer

Here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#1) are some reasons why you may be getting low transfection efficiency, along with suggested solutions.

Answer Id: E8981

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Product FAQ

Why do I see precipitates on the cells after transfection?

Answer

A small granular-like precipitate may be detected microscopically on the cells after transfection using cationic lipid. This is normal. The presence or absence of this precipitate is not indicative of the transfection efficiency. The precipitate can be caused by presence of excess EDTA or cationic lipid. Use DNA in water or, if in TE, use EDTA concentrations of <0.3 mM in the diluted DNA. Ensure concentrations of cationic lipid reagents do not exceed recommended amounts in complex formation.

Answer Id: E3996

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Product FAQ

Are lipid transfection reagents fluorescent?

Answer

The intrinsic fluorescence of Lipofectamine™ 2000 and Lipofectamine™ under FITC conditions was examined and the results were as follows:
- Lipofectamine™ 2000 in serum-free conditions (with or without DNA) - weak whitish fluorescence.
- Lipofectamine™ 2000 in serum-containing conditions (with or without DNA) - medium white and red fluorescence.
- Lipofectamine™ & Lipofectamine™ PLUS (with or without DNA) - orange fluorescence.
- We haven't seen any significant green fluorescence attributable to the Lipofectamine™.

These observations were made after transfection of CHO-K1 cells and by examining the live cells in PBS. Other lipid transfection reagents were not studied extensively on this issue, but it is very likely they will produce fluorescence in transfected cells.

Fluorescence from various media components may be where the problem lies. For example, riboflavin apparently fluoresces in the green wavelength and thus DMEM, which has a high riboflavin content, can be problematic in fluorescence experiments. Ham's F12 medium is 10x lower in riboflavin and is more suitable for fluorescence work.

One possible solution is to perform fluorescence imaging in PBS, rather than in culture medium. Also, check to make sure the cells are healthy and intact. Lysed cells or cells under stress may generate autofluorescent products.

Answer Id: E4451

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