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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

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 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

Why do I see cytotoxicity after performing transfection? Can you please help?

Answer

Here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#2) are possible causes for reduced viability following transfection, along with suggested solutions. Please note that as per recent findings, antibiotics can be used in media 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 stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.

Answer Id: E8982

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Product FAQ

I am working with well sizes different from those specified in your protocol. How do I scale up or scale down my transfection reaction?

Answer

Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details.

Answer Id: E8964

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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

Why are my transfections not reproducible?

Answer

Please review these possible causes for non-reproducible transfections, along with suggested solutions (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#3). Also, we recommend setting up one master mix of the DNA/lipid complex for the number of transfections planned to reduce multiple pipetting errors. When adding your complexes, we recommend changing tips between wells since re-used tips could bring carryover, especially for the 96- or 384-well format with small-volume formats.

Answer Id: E8983

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Product FAQ

Why is my transfection not reproducible?

Answer

In general, transfection efficiency will show some degree of variability from one transfection to another, no matter how hard one tries to control the parameters of transfection. Keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. To minimize the effect of transfection variability, one can use an internal reference control such as beta-galactosidase or luciferase. Co-transfect the expression plasmid with the reference plasmid and assay for the activity of beta-gal or luciferase.

Answer Id: E8965

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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

Why would the expression level of my gene in transient transfectants be greater than that in stable transfectants?

Answer

Expression in transiently transfected clones is typically higher because transiently transfected cells have a higher copy number of the gene (hundreds per cell). Stably transfected clones usually harbor 1-2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.

Answer Id: E8966

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Product FAQ

Is there a place where I can find references from other researchers who have used your transfection reagents?

Answer

Visit the product page for each reagent type and you will see a list of references at the bottom of the page. A table that lists specific cell line references is also accessible. We also recommend www.highwire.org as a search engine to find a large selection of up-to-date research articles using our transfection products. Simply include the name of the transfection reagent and your cell line/application of interest in your search criteria.

Answer Id: E8999

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Product FAQ

Can I use Lipofectamine™ 3000 in RNAi experiments?

Answer

Lipofectamine™ 3000 works well for delivery of vector-based RNAi and synthetic siRNA, as well as co-transfection of siRNA with plasmid DNA. However, Lipofectamine™ RNAiMAX is our best transfection reagent for synthetic siRNA. For in vivo siRNA experiments, we recommend Invivofectamine™ 3.0 Reagent.

Answer Id: E9016

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Product FAQ

What is the shelf life of the lipid transfection reagents?

Answer

Stored at 4°C in a sealed container, the lipids are stable for 12 months. Do not freeze the lipids. At 4°C with long term storage, due to evaporation concentration of lipid may vary, please briefly spin the contents before use. Add less lipid if you start noticing toxicity.

Answer Id: E3132

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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

Where can I find cell line-specific transfection protocols?

Answer

Cell line-specific transfection protocols can be found here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html). If you do not find a cell line-specific protocol or if the protocol does not perform as expected, we recommend testing the conditions described in the protocol supplied with the product to determine the optimal protocol. Successful transfection depends on the cell type, amount of lipid, cell health, passage number, and cell density at the time of transfection. Each of these factors may differ slightly from lab to lab and may require additional optimization of the protocol to achieve the same result.

Answer Id: E8967

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