I've mistakenly frozen my lipid reagent-can I still use it?

Product FAQ

Answer

Freezing can alter the integrity and composition of the lipid particles. The performance of the reagent may be affected, and it may only work for the first week or so. It is safer to purchase a new vial to ensure function.

Answer Id: E9064

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Is Lipofectamine™ 3000 the same as Lipofectamine™ 2000? What about Lipofectamine™ LTX, Lipofectamine™ PLUS, and Lipofectin™?

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Answer

These are all different cationic-lipid formulations. Lipofectamine™ 3000 provides the best transfection performance for both plasmid DNA and RNAi delivery over the broadest range of cell types. Lipofectamine™ LTX was designed for delivery of plasmid DNA with minimal cytotoxicity. Lipofectamine™ PLUS is a discontinued transfection reagent, although the PLUS™ Reagent is available and sold separately (Cat. No. 11514-015). Lipofectin™ was originally launched in the late 1980s and is considered our very first transfection reagent. We continue to offer these products for customers who prefer the older formulations, but recommend that all new customers try Lipofectamine™ 3000 first for optimal performance and lowest toxicity.

Answer Id: E8976

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

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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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I accidentallly left my lipid reagent at room temperature. Can I still use it?

Product FAQ

Answer

Yes, all of our lipid transfection reagents are stable at room temperature for months.

Answer Id: E9065

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Can I use the same amount of any lipid reagent for transfection of my particular cell line?

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Answer

No. The amount of lipid for each lipid reagent should be optimized for each cell line. Each lipid reagent has different composition/formulation, which will have different impact on each cell line. Therefore please optimize whenever you have a new lipid for each cell line.

Answer Id: E3126

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What is the main difference between transient and stable transfection?

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Answer

In transient transfection, the transfected DNA does not integrate into the host genome, as a result of which the foreign DNA will be lost at a later stage when the cells undergo mitosis. The expression is short-lived (maximum of 7-10 days) but the level of expression is high, since several copies of the DNA are delivered into the cell. In stable transfection, the transfected DNA integrates into the host genome and therefore remains in the genome of the cells and their daughter cells. The expression is thus sustained as long as the selection pressure is maintained. The expression level is low since only 1-2 copies of the DNA are integrated per cell. Transfection efficiency in a stable transfection is about 1-10% of that in a transient transfection.

Answer Id: E8977

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Where can I find cell line-specific transfection protocols?

Product FAQ

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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What is the difference between reverse transfection and forward transfection? What should I use?

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Answer

In forward transfection, cells are plated in wells, and the transfection complex is generally prepared and added the next day. In reverse transfection, the transfection complexes are prepared inside the wells, after which cells and medium are added. Reverse transfection is faster to perform than forward transfection, and is the method of choice for high-throughput transfection. For non–high-throughput transfections, generally forward transfections have better efficiency for most cell types.

Answer Id: E9003

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What recommendations do you have for selecting a transfection reagent?

Product FAQ

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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Is my lipid (Lipofectin™, Lipofectamine™, DMRIE-C, Lipofectamine™ 2000, Oligofectamine™, Cellfectin™ II) supposed to be cloudy?

Product FAQ

Answer

It is normal to see some turbidity and cloudiness in DMRIE-C, Cellfectin™ II and Lipofectamine™ 2000, and the others should be clear. Lipid transfection reagents are sensitive to low temperature; if you put them at temperature below 4 °C they will precipitate or even freeze and lower the efficiency. Most lipids will go cloudy and precipitate upon freezing, and may not be active anymore.

Answer Id: E4000

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

Product FAQ

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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What is the main advantage of lipid-meditated transfection over calcium phosphate-mediated transfection?

Product FAQ

Answer

The main advantage of lipid-mediated transfection is the higher transfection efficiency that can be achieved with cell types that cannot be transfected using calcium phosphate. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.

Answer Id: E8978

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

Product FAQ

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: E8968

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

Product FAQ

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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How can you improve low transfection efficiency with Lipofectin™?

Product FAQ

Answer

Incubate Lipofectin™ Reagent in Opti-MEM™ I Medium (or other medium without serum) for 30 minutes prior to adding DNA to help improve the transfection efficiency up to 3-fold.

Answer Id: E4002

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