Documents & Support

Lipofectamine 3000 Transfection Reagent was specifically designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cells. It works well for plasmid DNA transfection, siRNA transfection, and co-transfection of plasmid DNA with siRNA.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

Antibiotics can be used in the medium for culturing of cell lines. However, we do not recommend using antibiotics in the transfection medium unless previously tested in the cell type and payload being transfected. This is because presence of antibiotics during transfection may adversely affect transfection efficiency (i.e., positively charged antibiotics binding to the DNA being transfected) and overall health of cells being transfected.

For stable transfection, we recommend waiting wait 24-48 hrs after transfection before adding selected antibiotics.

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Was this answer helpful?

Yes. High transfection efficiency can be achieved in stem cells in suspension with reverse transfection using Lipofectamine Stem Transfection Reagent. Stem cells can easily be transfected at the time of passaging and during re-plating, when they are in suspension. For simple instructions on using reverse transfection with Lipofectamine Stem Transfection Reagent, please take a look at the transfection protocols for pluripotent stem cells, listed under Documents, Product Literature on the product page (https://www.thermofisher.com/order/catalog/product/STEM00008?ICID=search-product).

Was this answer helpful?

Yes, Lipofectamine 3000 can be used to transfect primary cells including neurons, stem cells, and blood cells as shown here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html). For very difficult-to-transfect cells, we recommend the Neon Transfection System.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

In forward transfection, cells are seeded to appropriate confluence or cell density in wells or dishes, and the lipid-DNA complexes are 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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

Cell line-specific transfection protocols can be found here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/transfection-selection-tool.html). If you do not find a cell line-specific protocol or if the transfection does not perform as expected, we recommend optimizing the conditions described in the product manual. 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 may require additional optimization of the protocol to achieve the same result. Please review our helpful troubleshooting tips: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html. For more troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport).

Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.

Was this answer helpful?

In general, transfection efficiency will show some degree of variability between transfection experiments and among replicates in the same transfection experiment. For better reproducibility, keep all transfection parameters, such as cell confluency, passage number, and phase of growth, consistent between transfections. If possible, thaw fresh cells. We recommend preparing one master mix of the DNA/lipid complexes 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. To further minimize the effects of transfection variability on data analysis, consider co-transfecting an internal normalization reference control such as beta-galactosidase or luciferase with the expression plasmid. Below are possible reasons for why your transfection results are not reproducible, along with suggested solutions:

1. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
2. Possible Cause: Transfections performed at different cell confluencies, or at different DNA:transfection reagent ratios
   Suggested Solution: Transfection performance reproducibility is dependent on day-to-day consistency in cell splitting, plating and transfecting with a consistent protocol (same DNA:transfection reagent ratios). Different DNA preparations or media changes may also change transfection performance.

Was this answer helpful?

In general, electroporation is a size-dependent transfection technique and transfection efficiency declines as plasmid size increases. We routinely use plasmids of 4-7 kb in our laboratories, and plasmids up to approximately 20 kb should not be a problem. Using plasmids larger than this will most likely result in lower transfection efficiency. Preliminary results indicate that bacterial artificial chromosomes (BACs) can be transfected as well, but with a low transfection efficiency. Keep in mind that in terms of molarity, 1 mg of a 6 kb plasmid corresponds to 2 mg of a 12 kb plasmid. This is why plasmid size is taken into consideration when comparing transfection efficiencies between plasmids of different lengths. For example: when comparing the transfection efficiency between 1 mg of a 10 kb plasmid and the transfection efficiency of a 150 kb BAC, 15 mg of the BAC would have to be used. This is not feasible since DNA amounts that large will cause cytotoxicity. On the other hand, this does not mean that BACs cannot be transfected using the Neon system. However, transfection efficiencies with a large amount of DNA will be very low.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

For transfecting of the Cas9 protein, we would recommend using the Neon transfection system or Lipofectamine CRISPRMAX Cas9 Transfection Reagent. For transfection of mRNA, we would recommend using Lipofectamine MessengerMAX Transfection Reagent. For transfection of CRISPR vectors, we would recommend using Lipofectamine 3000 Transfection reagent.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

Yes, Lipofectamine MessengerMAX Transfection Reagent is our best reagent for mRNA delivery and shows up to 5Xs the transfection efficiency of DNA transfection reagents in neurons and primary cells.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

The Neon Transfection System delivers DNA, mRNA, siRNA, and proteins efficiently into all mammalian cell types and is the best option when lipid-based transfection efficiencies are low. For immune or blood-derived cells, we generally recommend the Neon Transfection System. For neuronal cells, we recommend mRNA delivery with Lipofectamine MessengerMAX Transfection Reagent. For all stem cells, we recommend Lipofectamine Stem Transfection Reagent. Please visit our Transfection Reagent Selection Guide: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html Lentiviruses are also very efficient in the delivery of vectors for all cell types for gene expression and RNAi applications. We recommend producing high titer lentivirus particles using our LV-MAX Lentiviral Production System.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

Our cationic lipid transfection reagents are used to transfect DNA (plasmids or oligonucleotides), siRNA (or miRNA), mRNA, or proteins. DNA delivered may be in the form of plasmids, cosmids, or even YAC clones as large as 600 Kb. Please refer to the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to choose the best reagent based on cell type and application.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

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.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

Yes, transfection with mRNA results in faster and more immediate translation of protein and therefore, faster expression. We visually see expression of GFP in some cell lines as fast as 90 minutes after transfection. Additionally, transfection of mRNA with Lipofectamine MessengerMAX Reagent also provides prolonged duration of expression (GFP expression lasting for 5 days post-transfection), due to its ability to protect mRNA from degradation during transfection.

Find additional tips, troubleshooting help, and resources within our Transfection Support Center.

Was this answer helpful?

To study the effect of siRNA knockdown of the gene that is expressed by the co-transfected plasmid, both the plasmid DNA and siRNA can be co-transfected using Lipofectamine 3000 with the P3000 enhancer.

For co-transfection of plasmid DNA and siRNA, we recommend referring to this table (https://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_scaling.pdf) and using the amounts of plasmid DNA and siRNA suggested for individual transfection. For example, for co-transfection of plasmid DNA and siRNA in a 24-well plate, we would recommend using:
Plasmid DNA: 0.5 µg
siRNA: 15 pmol
Lipofectamine 3000 Reagent: 1.5 µL
P3000 Enhancer: 1 µL

Please note that the P3000 Enhancer is added to the co-transfection mix in order to promote the transfection of plasmid DNA. It has no effect on the transfection of siRNA and hence is not added to the transfection mix for an individual siRNA transfection.

Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

Was this answer helpful?