Cell transfection in a 24well plate using crisprmax. Transfection of mcf7 with lipofectamine 2000 maartje vogel. To perform transfection experiments in other cell culture plates, simply multiply the. Highest transfection efficiency in many cell types and formats e.
Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. Transfection guide overview of transfection methods. Genetic transfection an overview sciencedirect topics. 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.
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. 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. Optimized conditions for successful transfection of human. Use the procedure on below to transfect cells with plasmid dna. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Prewarm 50ml of optimum stored in cold room at 4c 10min.
To transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000, nucleic acid, cells. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids. Lipofectamine rnaimax has a broad peak of activity. The trusted, simple, broadspectrum transfection reagent for most cell lines. Each tube gets 8mls of optimem, 32ug of gag pol, 12. 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. Refer to the cell lines database for a list of cell types successfully. Invitrogen lipofectamine 2000 transfection reagent fisher. Mammalian cell penetration, sirna transfection, and dna.
Generally, transfection optimization could be achieved by transfecting cells with sirnas. We found that the trastuzumabresistant cell lines skbr3tr and bt474tr had low expression of mir126 and increased ability to migrate and invade. Cell transfection protocol for lipofectamine 2000 transfection of a 35 mm dish of 80% confluent cho cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1.
Caco2 transfection reagent colon carcinoma cells altogen. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Pdf optimization of sw480 colon cancer cells transfection. 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. Transfection guide overview of transfection methods promega.
Systematic screening of commonly used commercial transfection. 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 efficiencies lower than 50% were not reported in this table. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
The optimal volume of lipofectamine and quantity of plasmid. Lipofectamine transfection of c2c12 cells bridges lab. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. This protocol is well adjusted for cancer cell lines. Lipofectamine 3000 transfection reagent hepg2 liver. 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. 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. Maintain p19 cells undifferentiated in mem with 10% serum 7. Pdf optimization of kyse30 esophagus cancer cell line. Add 6l of lipofectamine2000 from biostores and 100 l of. 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.
For additional information, and protocols on transfecting suspension cells refer to the manual at. Cell culture media we use dmem supplemented with 10% fbs and 1% penstrep and passaging reagents pbs, trypsin micropipette and tips 1. B hela cells were transfected with luciferase encoding plasmid dna using either transit. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. 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. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. The resistance, invasion and mobilization abilities of the cells resistant to trastuzumab were reduced by ectopic expression of mir126 mimics. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. This allows the dna and the lipofectamine to form the complexes that will get the dna into the cells.
Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. 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. Transfecting sirna into mammalian cells using lipofectaminez 2000, continued transfection procedure use the following procedure to transfect mammalian cells in a 24well format. Plate cells in a 35mm dish at least a day before transfection b. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. Plate cells so they will be 7090% confluent at the time of transfection. The most effective transfection results were obtained with hela, huh7, modek and mg63 cell lines with both nanoparticles and lipofectamine. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent. Higher densities can lead to spontaneous differentiation. Lipofectamine was the most efficient transfection agent, but the efficiency of the nanoparticles was typically close to lipofectamine. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng.
Lipofectamine 2000 volumes on cell transfection efficiency was evaluated. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Lipofectmax transfection reagent has been tested to work the same efficiency as lipofectamine 2000 reagent, and used for the transfection of both. 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. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. 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. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Invitrogen lipofectamine 2000 transfection reagent life.
Invitrogen lipofectamine 2000 transfection reagent 0. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. 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. 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. 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. 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. Other reagents did not demonstrate effective transfection to this cell line. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Pdf optimization of sw480 colon cancer cells transfection with. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. 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.
However with the variability of primary cell types, there are challenges for efficient delivery of dna or rna. Lipofectamine 2000 transfection reagent dartmouth college. Improved delivery of cas9 proteingrna complexes using. Gently mix media on cells via figure 8 pattern and back and forth motions. To transfect cells with sirna, follow the protocol as. 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.
Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Lipofectamine 2000 dna transfection reagent protocol. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. 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.
I use 24well plates for transfections in triplicates. 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. Transfection protocol transfection components component cat. As always, use good cell culture practices and wear your personal protective. The cell numbers required for the transfection depend strongly on the growth rate and size of the cells. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Invitrogen lipofectamine 2000 transfection reagent. Transfecting sirna into mammalian cells using lipofectaminez 2000. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature.
Genscript recommends using lipofectamine 2000 for all transfections. 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. Do not add antibiotics to media during transfection as this causes cell death. The reagent provides high transfection efficiency in many cells types and formats. Transfection was measured by luciferase activity using a conventional assay. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. 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. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. As always, use good cell culture practices and wear your personal protective equipment.
Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high. A sample protocol is listed here for transfection experiments performed in 6well plates. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Combine the diluted dna and diluted lipofectamine, mix gently, and let incubate at room temperature for 1540 min.
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