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

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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 types of molecules can be transfected with cationic lipid reagents?

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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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Can antibiotics be used in media during transfection?

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

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

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

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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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What is the shelf life of the lipid transfection reagents?

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

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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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How does the use of Lipofectamine™ PLUS™ reagent improve transfection over other cationic lipid reagents?

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In general, the transfection activity peak with Lipofectamine™ PLUS™ reagent is broader, thus essentially eliminating the need for lengthy optimization experiments. Because the protocol includes a pre-complexing step with PLUS™ reagent, only half the amount of Lipofectamine™ reagent is required. The transfection time is short and the transfection can be done in the presence of serum. Difficult to transform cells (e.g. HT29, SKBR3 and MDCK) are transformed at a higher efficiency. See, FOCUS 19.3, 52.

Answer Id: E3163

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Do mammalian cells in culture divide in the presence of G418?

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Yes, cells can continue to divide up to two times in the presence of lethal doses of G418. The effect of the drug usually becomes apparent by two days.

Answer Id: E8969

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Can I use the PLUS™ Reagent in conjunction with Lipofectamine™ RNAiMAX?

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The PLUS™ Reagent will not enhance transfection efficiency when used in conjunction with Lipofectamine™ RNAiMAX.

Answer Id: E9040

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Can lipid reagents be used to cotransfect plasmids?

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Yes. The standard transfection protocol may be followed by keeping the total amount of DNA in the mixture constant. That is, if your protocol requires 1 ug plasmid, use 0.5 ug of each of two cotransfecting plasmids, or 0.25 ug of each of 4, etc. When performing cotransfections to introduce a selectable marker on a different plasmid, we recommend using a 3:1 to 10:1 molar excess of the plasmid of interest over the selectable plasmid to ensure that the plasmid of interest is present with the selectable plasmid.

Answer Id: E3133

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What is the main difference between transient and stable transfection?

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

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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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Which lipid transfection reagent do you recommend for my cell line?

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Answer

The best transfecting agent and efficiency would depend on the particular cell line you have. Please visit our online selection tools to see our recommendation for your specific cell line. If your cell line is not on the list, we recommend you try Lipofectamine™ LTX or Lipofectamine™ 2000 for plasmid transfection, and Lipofectamine™ RNAiMAX for siRNA transfection. For primary cells, Lipofectamine™ LTX with PLUS™ reagent is generally the best choice for plasmid transfection. For some hard-to-transfect cells, like suspension cells and stem cells, the Neon™ electroporation system usually works better compared to lipid transfection reagent.

Answer Id: E4502

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