To perform transfection experiments in other cell culture plates, simply multiply the. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine transfection of c2c12 cells bridges lab. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Refer to the cell lines database for a list of cell types successfully. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Other reagents did not demonstrate effective transfection to this cell line. For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high. The trusted, simple, broadspectrum transfection reagent for most cell lines. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. To transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000, nucleic acid, cells.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Lipofectamine was the most efficient transfection agent, but the efficiency of the nanoparticles was typically close to lipofectamine. Plate cells in a 35mm dish at least a day before transfection b. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. Gently mix media on cells via figure 8 pattern and back and forth motions. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.
However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. The reagent provides high transfection efficiency in many cells types and formats. Maintain p19 cells undifferentiated in mem with 10% serum 7. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent. Lipofectmax transfection reagent is a lipidbased transfection reagent that forms a complex with dna or rna, and transports the complex into a variety of adherent and suspension cell lines. Cell transfection in a 24well plate using crisprmax. If you wish to transfect cells in other tissue culture formats, you will need to determine the optimal conditions to use for your mammalian cell line. The insertion of dna into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of rna into a cell is used to downregulate the production of a specific protein by stopping translation. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. As always, use good cell culture practices and wear your personal protective. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Pdf optimization of sw480 colon cancer cells transfection. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11.
Lipofectmax transfection reagent has been tested to work the same efficiency as lipofectamine 2000 reagent, and used for the transfection of both. A polymerbased transfection caco2 kit for colon adenocarcinoma cells yields at least 94% transfection efficiency and is a helpful tool for gene expression studies as well as for assessing cellular functionality. Transfecting sirna into mammalian cells using lipofectaminez 2000, continued transfection procedure use the following procedure to transfect mammalian cells in a 24well format. Invitrogen lipofectamine 2000 transfection reagent fisher. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. I use 24well plates for transfections in triplicates. Invitrogen lipofectamine 2000 transfection reagent 1. Transfection efficiencies lower than 50% were not reported in this table. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Caco2 transfection reagent colon carcinoma cells altogen. While immortalized cell lines traditionally have served as the common targets of transfection, primary cells are often preferred as they more closely resemble the invivo nature of tissues.
Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectamine 2000 volumes on cell transfection efficiency was evaluated. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Before you start akin to forward transfection, optimizing transfection conditions by transfecting a reporter plasmid into the cell type of interest such as a luciferase or gfp encoding plasmid. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2.
Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. The most effective transfection results were obtained with hela, huh7, modek and mg63 cell lines with both nanoparticles and lipofectamine. This protocol is well adjusted for cancer cell lines. Mouse myeloma cell lines sp20, ns0, ns1, ag8, and p3u1 were transfected with pegfpn1 vector using lipofectamine 2000, jetpei and lyovec commercial transfection reagents. Transfection protocol transfection components component cat. Improved delivery of cas9 proteingrna complexes using. Lipofectamine 2000 reagent thermo fisher scientific. Genscript recommends using lipofectamine 2000 for all transfections. Transfection was measured by luciferase activity using a conventional assay. Cell transfection protocol for lipofectamine 2000 transfection of a 35 mm dish of 80% confluent cho cells. Each tube gets 8mls of optimem, 32ug of gag pol, 12. Generally, transfection optimization could be achieved by transfecting cells with sirnas. Do not add antibiotics to media during transfection as this causes cell death.
Mouse myeloma cell lines sp20, ns0, ns1, ag8, and p3u1 were transfected with pegfpn1 vector using lipofectamine 2000, jetpei and lyovec commercial transfection reagents in different combinations. Invitrogen lipofectamine 2000 transfection reagent 0. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids high transfection rates for adherent mammalian cell lines and primary cell cultures suspension cells and cells from other organisms also successfully transfected but need to be immobilized mild treatment of cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. Add 6l of lipofectamine2000 from biostores and 100 l of. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. The cell numbers required for the transfection depend strongly on the growth rate and size of the cells. Lipofectamine rnaimax has a broad peak of activity. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Invitrogen lipofectamine 2000 transfection reagent.
Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. As always, use good cell culture practices and wear your personal protective equipment. In contrast to the efficiency of gene suppression in hela cells, treatment with lipofectamine 2000 and 50 nm sirna in imcd, 3t3l, pc12, and jurkat cells effected no significant decrease in gapdh protein levels fig. This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Invitrogen lipofectamine 2000 transfection reagent life. Plate cells so they will be 7090% confluent at the time of transfection. A sample protocol is listed here for transfection experiments performed in 6well plates. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25.
Cell culture media we use dmem supplemented with 10% fbs and 1% penstrep and passaging reagents pbs, trypsin micropipette and tips 1. However with the variability of primary cell types, there are challenges for efficient delivery of dna or rna. We found that the trastuzumabresistant cell lines skbr3tr and bt474tr had low expression of mir126 and increased ability to migrate and invade. Genetic transfection an overview sciencedirect topics. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids. Systematic screening of commonly used commercial transfection. Lipofectamine 2000 dna transfection reagent protocol. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng. To transfect cells with sirna, follow the protocol as. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids high transfection rates for adherent mammalian cell lines and primary cell cultures suspension cells and cells from other organisms also successfully transfected but.
Transfection guide overview of transfection methods promega. Transfecting sirna into mammalian cells using lipofectaminez 2000. Optimized conditions for successful transfection of human. Transfection guide overview of transfection methods. Transfection of mcf7 with lipofectamine 2000 maartje vogel. The optimal volume of lipofectamine and quantity of plasmid. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Higher densities can lead to spontaneous differentiation. Combine the diluted dna and diluted lipofectamine, mix gently, and let incubate at room temperature for 1540 min. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages.
Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. Use the procedure on below to transfect cells with plasmid dna. Mammalian cell penetration, sirna transfection, and dna. Pdf optimization of sw480 colon cancer cells transfection with. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using invitrogen lipofectamine 3000 transfection reagent. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Lipofectamine 3000 transfection reagent hepg2 liver. Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent.
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