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11514015

Product FAQ

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

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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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

Which lipid transfection reagent would you recommend trying for my cell line?

Answer

We recommend using Lipofectamine™ 3000 Reagent or delivery of plasmid DNA, Lipofectamine™ MessengerMAX™ Reagent for delivery of mRNA or short oligos, and Lipofectamine™ RNAiMAX Reagent for delivery of siRNA/miRNA.

Answer Id: E8993

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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

How do I perform a dose-response curve or kill curve?

Answer

The dose-response curve is a valuable tool to determine cell toxicity when exposed to various concentrations of antibiotic. The amount of selective antibiotic required to select for resistant cells varies with a number of factors, including cell type and type of antibiotic. We recommend performing a dose-response curve every time a new antibiotic (or a different brand) or a different cell line is used.

Experimental outline of dose-response curve assay:

1.Plate cells in a number of wells such that they are 25–30% confluent. This means that the cells are still dividing and hence will respond well to the antibiotic.
2.Dilute the antibiotic being tested to a broad linear concentration of the recommended range in growth medium.
3.Remove the growth medium from the cells. Apply the antibiotic-containing medium to the respective wells, leaving one set of wells empty. To these wells, add growth medium that does not contain the antibiotic.
4.Culture cells under proper growth conditions (change the medium every 3–4 days to get rid of dead cells and add fresh medium containing antibiotic) and observe the cells daily. At 10–14 days, assess the number of viable cells in each well. (This time period depends upon the antibiotic being tested; antibiotics such as Geneticin™, Hygromycin, and Zeocin™ take about 3 weeks to kill cells, so waiting for 10–14 days would be ideal. However, for Blasticidin, which kills cells in about 2 weeks, waiting for 7–10 days would be sufficient.) To do this, aspirate the medium, wash the cells with phosphate-buffered saline and stain the cells with 0.5% methylene blue and 50% methanol for 20 minutes.
5.Plot the number of viable cells against the antibiotic concentration. This curve is the dose-response curve or kill curve. The lowest concentration of the antibiotic that kills all the cells in the chosen time period is then used for the stable selection.

Answer Id: E8980

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Product FAQ

I have tried several different transfection reagents and have failed to transfect my gene into my cell line of interest. Do you have any suggestions?

Answer

We recommend that you try electroporation as a method of delivering your plasmid of interest. We offer the Neon™ Transfection System for highly efficient transfection of primary cells, stem cells, and difficult-to-transfect cells. You may also consider using a viral-based system (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/viral-delivery-mammalian-expression.html) to deliver your gene into your mammalian cell line of interest.

Answer Id: E8985

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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

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

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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Product FAQ

What is the PLUS™ Reagent?

Answer

PLUS™ Reagent is a proprietary reagent for pre-complexing DNA. It enhances cationic lipid-mediated transfection of DNA into many cultured eukaryotic cells when used in conjunction with a transfection reagent. Use PLUS™ Reagent to enhance transfection results with Lipofectamine™ Reagent, Lipofectamine™ LTX Reagent, Lipofectin™ Reagent, Cellfectin™ Reagent, and Oligofectamine™ Reagent. Lipofectamine™ LTX is offered in combination with the PLUS™ Reagent in Cat. Nos. A12621, 15338030, and 15338100. PLUS™ Reagent is also offered as a stand-alone product (Cat. No. 11514015). We do not recommend using the PLUS™ Reagent with Lipofectamine™ 2000 or Lipofectamine™ 3000.

Answer Id: E9026

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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

What points should I consider to achieve optimal transfection with a lipid-based transfection reagent?

Answer

Here are some points to consider:

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 that the promoter-enhancer of the transfected DNA is compatible with the target cell type.
7. Do not use a cationic lipid reagents 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: E8989

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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

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

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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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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