How can I improve transfection efficiency with cationic lipids?

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Answer

1. Select the cationic lipid reagent that is likely to result in highest transfection efficiency for your cell type. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to make the right choice.
2. Optimize the cationic lipid reagent and DNA amounts. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum may contain components that could interfere with complex formation. We recommend using Opti-MEM™ I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, keeping in mind that the efficiency of complex formation may not be as high as with Opti-MEM™ I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be from 50% to 80% confluency at the time of transfection (for Lipofectamine™ 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase. For better consistency of results between transfection experiments, it would be best to accurately count your cells with a hemocytometer or with the Countess™ II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Make sure the promoter-enhancer of the transfected DNA is compatible with the target cell type.
7. Do not use cationic lipid reagent that has been frozen or stored in a section of the refrigerator where the temperature is below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

Also, please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Answer Id: E3994

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Why do I see precipitates on the cells after transfection?

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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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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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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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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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How can you improve low transfection efficiency with Lipofectin™?

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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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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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Can lipid reagents be used to cotransfect plasmids?

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Answer

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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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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I don't see a cell line-specific transfection protocol. What conditions should I try?

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Answer

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 require additional optimization of the protocol to achieve the same result.

Answer Id: E8992

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What is the stability of your transfection reagents?

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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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What kind of tubes should I use for making lipid:DNA complexes?

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Answer

Polypropylene, polystyrene, or glass tubes may be used with any of our transfection products without issue.

Answer Id: E8996

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

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

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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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Can you compare and contrast the various cationic lipid-based transfection reagents you offer in terms of composition and application?

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Answer

- Lipofectamine™ 3000 & P3000™ Reagents are of a proprietary formulation.
- Lipofectamine™ 2000 Reagent is a proprietary formulation.
- Lipofectamine™ MessengerMAX Reagent is a proprietary formulation.
- Lipofectamine™ RNAiMAX Reagent is a proprietary formulation.
- Lipofectamine™ LTX Reagent is a proprietary formulation.
- Lipofectin™ Reagent is a 1:1 (w/w) formulation of N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA) and the neutral lipid dioleolyphosphatidylethanolamine (DOPE).
- Lipofectamine™ Reagent is a 3:1 (w/w) formulation of the polycationic lipid 2.3-dioleyloxy-N(2(sperminecarboxamido)ethyl)-N, N-dimethyl- 1-propanaminium trifluoroacetate (DOSPA), and DOPE.
- PLUS™ Reagent is a proprietary formulation.
- DMRIE-C Reagent is a 1:1 (M/M) liposome formulation of the cationic lipid DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and cholesterol.
- Cellfectin™ II Reagent is a proprietary formulation.

- Lipofectamine™ 3000 Reagent results in the highest efficiency of plasmid DNA delivery into most cell types. It also works well for siRNA transfection and co-transfection of plasmid DNA with siRNA.
- Lipofectamine™ 2000 Reagent works well with most adherent cells as well as difficult-to-transfect cells. It can be used for transfection of plasmid DNA, siRNA, and co-transfection of plasmid DNA with siRNA.
- Lipofectamine™ MessengerMAX Reagent is recommended for transfection of mRNA or short oligos.
- Lipofectamine™ RNAiMAX provides the best results when transfecting siRNA/miRNA.
- Cellfectin™ II Reagent is recommended for transfection of insect cells.

Answer Id: E9005

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