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View additional product information for Lipofectamine™ 3000 Transfection Reagent - FAQs (L3000015, L3000001, L3000008, L3000150, L3000075)
58 product FAQs found
Yes, Lipofectamine 3000 Transfection Reagent is stable at room temperature for months.
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
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 use a new vial.
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
Yes, all of our lipid transfection reagents are stable at room temperature for months.
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
While Lipofectamine 3000 or Lipofectamine 2000 may be used for co-transfection of siRNA with plasmid DNA, Lipofectamine RNAiMAX cannot be used.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Yes, Lipofectamine MessengerMAX Transfection Reagent can deliver plasmid DNA in combination with mRNA (i.e., in CRISPR); however, Lipofectamine 3000 Transfection Reagent is best optimized for plasmid DNA delivery and superior DNA transfection performance.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
We recommend using Lipofectamine 3000 which demonstrates much higher transfection efficiency and protein expression over all other transfection reagents for suspension, hard-to-transfect, and primary cells. Review Lipofectamine 3000 data for over 60 cell lines here https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html. For highest transfection efficiencies, we recommend the Neon Transfection System for Jurkat (94%) and K562 (90%) cells.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
We have tested four primary cell lines: HuVEC (human ubilical vein endothelial cells), NHFF (normal human foreskin fibroblast), MJ90, and NDHF with transfection efficiencies between 5-25%. Instead, we recommend using Lipofectamine 3000 which shows highest transfection efficiency for all cell types and optimize by varying the DNA and lipid ratio. Alternatively, the Neon Transfection System shows 75-80% transfection efficiency. Please visit our Neon cell specific protocol database for more information: https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html?icid=fr-neon-3
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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.
For transfection of standard non-modified siRNA, Silencer siRNA, Stealth RNAi siRNA, Silencer Select siRNA, Invitrogen Pre-miR Precursors, mirVana miRNA Mimics, Invitrogen Anti-miR Inhibitors, and mirVana miRNA Inhibitors, use only the Lipofectamine 3000 Reagent for efficient delivery to the cytoplasm. There is no need to use the P3000 Reagent.
For transfection of plasmid DNA, vector-based BLOCK-iT shRNA or miRNA, ViraPower HiPerform lentiviral expression vectors, or GeneArt CRISPR nuclease vectors, use Lipofectamine 3000 reagent with the P3000 Reagent for efficient delivery to the nucleus.
Please refer to the manual (https://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_protocol.pdf) and to this scaling table (https://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_scaling.pdf) for guidelines on amounts to use to prepare the transfection mixes.
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
Lipofectamine 3000 works well for delivery of vector-based RNAi and synthetic siRNA, as well as co-transfection of siRNA with plasmid DNA. However, Lipofectamine RNAiMAX is our best transfection reagent for synthetic siRNA. For in vivo siRNA experiments, we recommend Invivofectamine 3.0 Reagent.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
The Lipofectamine 3000 optimization protocol in the manual may be used for all cell types. However, we offer a growing number of relevant cancer cell line-specific protocols at the following link: https://www.thermofisher.com/order/catalog/product/L3000015?ICID=search-product
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
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.
You can use Lipofectamine 3000 for transfection of suspension cells, hard-to-transfect cells, and primary cells. Please see data here (https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html). However, if you experience low transfection efficiency, we recommend the Neon Transfection System or the ViraPower Lentiviral Delivery Systems.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Yes, Lipofectamine 3000 Transfection Reagent is an animal origin-free (AOF) product.
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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.
We recommend using Lipofectamine Stem Transfection Reagent for delivery of DNA, mRNA and co-transfection (siRNA and plasmid DNA) in a wide variety of stem cells. Lipofectamine Stem Transfection Reagent also delivers Cas9-gRNA ribonucleoproteins for gene editing applications. For more information on the transfection efficiency and versatility of the reagent, please visit: https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-stem-transfection-reagent.html
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
For getting successful co-transfection of plasmid DNA and siRNA, we recommend using high-quality DNA, a validated siRNA against the gene of interest, and log-phase growing cells. The method or application for co-transfection is important to help you determine the order of delivery and the right reagent for transfection:
1. 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 the amounts of plasmid DNA, siRNA, Lipofectamine 3000, and P3000 Enhancer to use for co-transfection, please refer to the manual (http://tools.thermofisher.com/content/sfs/manuals/lipofectamine3000_protocol.pdf) and use the same amounts as recommended for individual transfection of plasmid DNA and siRNA.
2. To study the effect of siRNA knockdown of an endogenous gene, we recommend transfecting the siRNA first using Lipofectamine RNAiMAX. 4-48 hours after delivery of siRNA, the plasmid DNA can be transfected using Lipofectamine 3000. The time of post-transfection delivery of plasmid DNA may need to be optimized based on the half-life of the protein to be knocked down.
Once the appropriate method of delivery is determined based on the application, transfection should be optimized for DNA, siRNA, and transfection reagent doses. For transfecting HEK293 cells in 96 wells, we typically use these amounts per well:
- DNA: 0.1-0.2 µg
- siRNA: 1-3 pmoles
- Lipofectamine 3000: 0.1-0.3 µL
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
Lipofectamine 3000 (Cat. No. L3000015is our best transfection reagent and we highly recommend this over all other reagents for plasmid DNA delivery for a broad range of cell types which includes:
- 60 cell lines (i.e., cancer, fibroblasts, neural, etc.)
- Easy-to-transfect cells
- Hard-to-transfect cells (i.e., neural, stem, blood-derived)
- Suspension cells
- Primary cells
- iPSCs
Please see our table of validated cell lines (https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html).
Lipofectamine 2000 (Cat. No. 11668019) and Lipofectamine LTX (Cat. No. 15338100) are alternative options that may work better for certain cell types. For addressing cytotoxicity issues, Lipofectamine 3000 is our most gentle reagent with significantly reduced cytotoxicity levels and improved transfection performance. Both Lipofectamine 3000 and Lipofectamine 2000 may be used for co-transfection (siRNA and plasmid DNA) experiments.
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
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.
We recommend using Lipofectamine RNAiMAX Reagent (Cat. No. 13778150) for delivery of siRNA and miRNA 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 (Cat. No. L3000015) with the P3000 Enhancer Reagent. For CRISPR-mediated gene knockout methods, we recommend Lipofectamine CRISPRMAX (Cat. No. CMAX00015) for Cas9/gRNA ribonucleoprotein, Lipofectamine MessengerMAX (Cat. No. LMRNA001) for Cas9 mRNA, and Lipofectamine 3000 (Cat. No. L3000015) with the P3000 Enhancer Reagent for CRISPR plasmid DNA-based delivery. For all difficult-to-transfect cells, we recommend using electroporation methods such as the Neon Transfection System (Cat. No. MPK5000).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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.
It is not necessary to use serum-free medium during lipid transfection. However, it is critical 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 Transfection Reagent, we recommend performing transfection in medium without serum.
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
Polypropylene, polystyrene, or glass tubes may be used with any of our transfection products without issue.
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
We recommend using Lipofectamine 3000 Reagent (Cat. No. L3000015) for the delivery of plasmid DNA, Lipofectamine MessengerMAX Reagent (Cat. No. LMRNA001) for mRNA or short oligos, and Lipofectamine RNAiMAX Reagent (Cat. No. 13778150) for siRNA or miRNA. 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.
To ensure optimal transfection success, we recommend including a positive transfection control and additional controls to confirm cell health and reagent quality.
For DNA transfection, we recommend using the pJTI R4 Exp CMV EmGFP pA Vector (Cat. No. A14146). For siRNA transfection, we recommend using either the BLOCK-iT AlexaFluor Red Fluorescent Control (Cat. No. 14750100) or Silencer Select GAPDH Positive Control siRNA (Cat. No. 4390850). For protein transfection, we recommend co-transfecting with an EmGFP mRNA such as the Tri-Link CleanCap EGFP mRNA (Cat. No. L-7201).
Cell health and reagent quality controls:
- Cells only
- Cells + DNA or RNA or protein only
- Cells + lipid reagent only
- Cells + Opti-MEM only
- Cells + positive control
Find additional tips, troubleshooting help, and resources within ourTransfection Support Center.
Here are some points to consider:
1. Select the lipid reagent that is likely to result in highest transfection efficiency for your cell type, payload, and application. 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 choose the best reagent.
2. Optimize both lipid reagent and DNA quantities. 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, but 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 between 50% to 80% confluency at the time of transfection (please refer to specific reagent manual for details). Cells should be in the mid-log growth phase. For better consistency and reproducibility of results between transfection experiments, accurately count your cells with either a hemocytometer or the Countess II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Confirm that the promoter and/or enhancer (any gene regulatory sequences) of the transfected DNA is compatible with the target cell type.
7. Do not use a cationic lipid reagent that has been frozen or stored at temperatures 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).
For additional tips ,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).
Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.
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 shipment 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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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.
Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.
It is normal to see some precipitate on the cells. However, a common reason for detecting unsually high precipitate on cells following lipid-based transfection is if there is excess EDTA or cationic lipid present. We recommend diluting the DNA in water or, if TE is preferred, use EDTA concentrations of <0.3 mM. Also ensure that concentrations of cationic lipid reagents do not exceed recommended amounts during complex formation. The presence or absence of this precipitate is not indicative of the transfection performance.
Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.
Below are possible reasons why you may see reduced viability following transfection, along with suggested solution.
Below are possible reasons why you may be getting low transfection efficiency, along with suggested solutions:
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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Transfection does not work for certain cell types such as non-dividing cells, whereas viral transduction works for dividing as well as non-dividing cells, such as neuronal cells that are hard to transfect.
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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. Calcium phosphate is prone to variability due to its sensitivity to slight changes in pH, temperature, and buffer salt concentrations. Calcium phosphate may also be cytotoxic to many cell types, especially primary cells. Further, lipid-mediated transfection can be used to deliver DNA ranging from oligos to large DNA, and can also deliver RNA and protein.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
During transient transfection the exogenous DNA does not integrate into the host genome, as a result some DNA is lost with every subsequent cell division. The expression is short-lived (maximum of 7-10 days) but the level of expression is high, since up to hundreds of copies of the DNA may be delivered into the cell. In stable transfection, under antibiotic selection pressure, the DNA integrates into the host cell genome and is passed onto their daughter cells during cell division. The expression is thus sustained as long as the selection pressure is maintained. The expression level is low since only 1-2 copies of the DNA may be integrated per cell. Transfection efficiency in a stable transfection is about 1-10% of that in a transient transfection.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
All of our lipid reagents have different cationic-lipid formulations, each with unique properties and specific applications. Lipofectamine 3000 (Cat. No. L3000015) provides the best transfection performance with lowest cytotoxicity for plasmid DNA and RNAi delivery for all cell types. Lipofectamine LTX (Cat. No. 15338100) was formerly designed for delivery of plasmid DNA with minimal cytotoxicity. Lipofectamine PLUS is a discontinued transfection reagent, although the PLUS Reagent is available and sold separately (Cat. No. 11514-015). Lipofectin (Cat. No. 18292011) was originally launched in the late 1980s and is considered our very first transfection reagent. We continue to offer these products for customers who prefer the older formulations, but recommend that all new users try Lipofectamine 3000 first for optimal performance and lowest toxicity.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
1 A260 unit (double stranded DNA in H2O) = 50 mg/mL. The extinction coefficient will change if DNA is diluted in a buffer other than H2O. This will change the value indicated above.
Sample calculation:
Volume of plasmid DNA sample = 100 mL
Dilution (1/20) = 25 mL of the sample in 475 mL H2O
A260 of diluted sample = 0.65
Note: For optimal results, make sure OD values are within 0.1 and 1.0.
Concentration of plasmid DNA sample = 0.65 x 50 mg/mL x 20 (dilution factor) = 650 mg/mL
Amount of plasmid DNA in sample = 650 mg/mL x 0.1 mL (sample volume) = 65 mg
An A260/A280 value that is between 1.8 and 2.0 means that the plasmid DNA is pure. A260/A280 readings that are less than 1.8 indicate that the sample may be contaminated with aromatic products (i.e., phenol) or protein. Readouts greater than 2.0 suggest that the sample is contaminated with RNA.
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For transfection-grade plasmid DNA, use either a PureLink HiPure or PureLink Expi Plasmid Purification Kit with plasmid purity equivalent to 2X CsCl gradients. For endotoxin-free DNA (< 9.1 EU/µg) use a PureLink Expi Endotoxin-Free Kit (Maxi, Mega, or Giga). Endotoxin-free DNA is recommended for sensitive applications such as transfection of primary cells and research on gene therapy for plasmid vaccines. For more information, please click here: http://www.thermofisher.com/us/en/home/brands/product-brand/purelink.html.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Yes. You may co-transfect plasmids and siRNA using i) Lipofectamine 3000 together with P3000 or ii) Lipofectamine 2000.
Find additional tips, troubleshooting help, and resources within ourTransfection Basics Support Center.
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.
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 µg plasmid, use 0.5 µg of each of two co-transfected plasmids, or 0.25 µg of each of 4 co-transfected plasmids. When performing co-transfections 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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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.
Expression in transiently transfected clones is typically higher because transiently transfected cells can have up to hundreds of copies of the plasmid containing the gene of interest. 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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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:
Each of our transfection reagent protocols provides a table for scaling up or down transfections. Please consult the specific manual for details.
For well or plates sizes not listed in the scaling table, calculate the total surface area and estimate the -fold difference from the 24-well. Use this -fold difference to adjust for reagent volumes, payload quantities, and seeding densities.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
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. 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 additional troubleshooting tips, please visit our Transfection Support Center (thermofisher.com/transfectionsupport)
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Yes, cell density is an important parameter in influencing transfection efficiency. If the seeding density is too low, some cytoxicity may be observed. If the cell density is high, lower than expected transfection efficiency may be observed. Both issues may be easily resolved by either descreasing or increasing the quantity of complexes added to the culture. We recommend using Lipofectamine 3000 since it shows the best flexibility for variable seeding density without showing cytotoxicity issues and maintains high protein expression.
Lipofectamine 3000, Lipofectamine 2000, and Lipofectamine LTX/PLUS provide excellent transfection efficiencies at confluencies between 70 and 90%. Some toxicity may be observed at lower confluencies but may be alleviated by decreasing quantity of complexes or removing the complexes after 4-6 hours incubation and refreshing the media. 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.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Choose the best reagent by cell type and application by using the Transfection reagent selection guide (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
There are many transfection methods available to deliver plasmids, DNA fragments, oligos, siRNAs, mRNA, or proteins for a wide range of research and drug discovery applications. A review of the pros and cons of each technique is provided here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/gene-delivery-selection-guide.html).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
We do not have a ready-to-use reverse transfection protocol for Lipofectamine 3000 reagent.
We have a reverse transfection protocol for RNAiMAX reagent, on the RNAiMAX Reverse Transfections Lipofectamine page, that can be used as a starting point and adapted as needed for your protocol and reagent volumes.
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Lipofectamine 3000 reagent was developed to have no cytotoxic effect, and therefore there is no need to change the medium after a transfection. The transfection complexes should be made in a serum- and antibiotic-free medium, such as Opti-MEM medium, and can be added to the cells growing in their regular growth medium. There is no need to change the medium afterwards.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
No, the P3000 reagent cannot be ordered separately.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Just keep the total DNA amount the same (e.g., half of the amount for each plasmid, or any other suitable ratio). Everything else remains the same.
We do not have specific culture media volume recommendations for transfection with Lipofectamine 3000 Transfection Reagent, but the following link outlines standard culture media volumes for different plate formats: https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
The ranges in this reference table can be used to optimize your own transfections.
Yes, Lipofectamine 3000 Transfection Reagent is stable at room temperature for weeks to months.
Sorry, the P3000 Enhancer Reagent is only available bundled along with Lipofectamine 3000 Transfection Reagent (Cat. No. L3000015).
Depending on your optimization ratios between Lipofectamine 3000 Transfection Reagent and P3000 Enhancer Reagent, it is possible that one of the reagents will run out sooner than the other. We purposely designed the formulation such that regardless of your optimization ratio, you will receive a high number of reactions because of the superior efficiency of this product.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.