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Product FAQ

Which transfection reagent do you recommend for RNAi applications?

Answer

We recommend using Lipofectamine™ RNAiMAX Reagent for delivery of siRNA into all cell types. It has been specifically developed for siRNA transfection while providing high transfection efficiency with minimal cytotoxicity. As a result, less optimization is necessary. For vector DNA–based RNAi applications, we recommend Lipofectamine™ 3000 Reagent with the P3000™ Enhancer Reagent.

Answer Id: E9000

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Product FAQ

Is there a place where I can find references from other researchers who have used your 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: E8968

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Product FAQ

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

Answer

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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Product FAQ

I accidentally kept my Lipofectamine™ RNAiMAX Reagent at room temperature. Can I still use it?

Answer

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

Answer Id: E9073

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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

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

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

What types of molecules can be transfected using your lipid-based transfection reagents?

Answer

Our cationic lipid transfection reagents can be used to transfect DNA, siRNA, Stealth™ RNAi, mRNA, dicer-generated siRNA pools, or plasmids containing shRNA cassettes. Oligonucleotides, proteins, and RNA can also be transfected. The DNA can be plasmids, cosmids, or even YAC clones up to 600 kb.

Answer Id: E8971

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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

I’m seeing cell death after transfection using Lipofectamine™ RNAiMAX Reagent. What should I do?

Answer

Check to see if antibiotics were added to the medium during transfection, as this can cause cell death. Test serum-free media for compatibility with Lipofectamine™ RNAiMAX Reagent.

Answer Id: E9074

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Product FAQ

What kind of tubes can I use to form DNA:lipid complexes?

Answer

Polypropylene, polystyrene, or glass tubes may be used with any of our transfection products.

Answer Id: E3124

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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

What controls do you recommend that I use when performing a transfection?

Answer

In general, our recommended controls include:

- Cells only
- Cells + DNA or RNAi only
- Cells + lipid reagent only
- Cells + GFP plasmid positive control

These controls are important as they check cell health, reagent toxicity, and reagent function simultaneously.

Answer Id: E8990

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Product FAQ

What types of molecules can be transfected with cationic lipid reagents?

Answer

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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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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