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Product FAQ

Are there any guidelines for choosing a lipid transfection reagent?

Answer

It is best to optimize for your cells and application. Here are some basic guidelines:

- Lipofectamine LTX and PLUS Reagent: Minimal optimization, excellent efficiency with adherent eukaryotic cells DNA, difficult cell lines
- Lipofectamine Reagent: Adherent eukaryotic cells, DNA, oligonucleotides
- Lipofectin Reagent: Transfecting DNA in eukaryotic cell
- Cellfectin II: Insect cells
- DMRIE-C Reagent: DNA, RNA, suspension cells
- Oligofectamine: Oligonucleotides
- Lipofectamine RNAiMAX: siRNA, pre-miR, miRNA, anti-miR

NOTE: Please also visit our online Transfection Selection Tool to get specific recommendation for your cell line

Answer Id: E3079

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Product FAQ

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

Answer

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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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 am getting very low transfection efficiency. Can you please provide some troubleshooting tips?

Answer

Here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#1) are some reasons why you may be getting low transfection efficiency, along with suggested solutions.

Answer Id: E8981

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Product FAQ

Is the passage number of my cells important to consider when doing transfection?

Answer

In general, once optimal transfection conditions are determined for a given cell line, it is recommended that cells be passaged less than 20 times to maintain reproducible results. Thus immediately following the determination of optimal conditions, cells should be frozen down so that when the working stock approaches 20 passages, a new batch can be started from the frozen stock.

Answer Id: E3852

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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

How can you improve low transfection efficiency with Lipofectin?

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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Product FAQ

Do you offer a reagent for the transfection of endothelial cells?

Answer

We recommend using the Lipofectamine 3000 Reagent for transfection of endothelial cells.

Answer Id: E9063

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Product FAQ

What is the shelf life of the lipid transfection reagents?

Answer

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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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

I am working with well sizes different from those specified in your protocol. How do I scale up or scale down my transfection reaction?

Answer

Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details.

Answer Id: E8964

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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

I have optimized my conditions for a particular cell line but I seem to be getting inconsistent results with my transfection. What factors contribute to this inconsistency?

Answer

Cells from a different passage number may behave differently. Also, if cells were sitting at confluence prior to plating for transfection, they may not transfect efficiently. To minimize such inconsistencies, passage the cells while they are still growing exponentially. Actively dividing cells transfect better.

Answer Id: E3125

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Product FAQ

Why do I see cytotoxicity after performing transfection? Can you please help?

Answer

Here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html#2) are possible causes for reduced viability following transfection, along with suggested solutions. Please note that as per recent findings, antibiotics can be used in media during transfection. We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For stable transfections, wait at least 72 hours after transfection before adding selective antibiotics.

Answer Id: E8982

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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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