Invitrogen lipofectamine 2000 transfection reagent. Use the procedure on below to transfect cells with plasmid dna. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. To transfect cells with sirna, follow the protocol as. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate. Fast and efficient transfection of mouse embryonic stem. Incubate for 20 min at rt 6 after incubation, add optidnalipo mixture to cells dropwise. Genscript recommends using lipofectamine 2000 for all transfections. Highest transfection efficiency in many cell types and formats e. For each plasmid to be transfected, prepare a separate, sterile 1. We did not find substantial transfection efficiency variations in. For each transfection sample, prepare complexes as follows.
Lipofectamine ltx transfection reagent from invitrogen. Scale up or down transfections to transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000 cd reagent, dna, cells, and medium used in proportion to the relative surface area, as shown in the following table. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Transfection protocol for hpc7 using lipofectamine 2000 igem 2017. As expected, transfection efficiency for both reagents correlates with the amounts of dna used. 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 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Liposome lipidbased formulation has the least influence. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Researchers use lipofectamine 2000 reagent for plasmid dna transfection as well as sirna and shrnabased gene knockdown. Descriptionlipofectamine 2000 is a proprietary formulation suitable for the transfection of. Lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine stem reagent was designed specifically for stem cells and confers low cytotoxicity, enabling researchers to achieve optimal. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Dnaintm transfection reagent family a comparative study of transfection reagents. Add 250 ul optilipo 2000 mixture to each tube with dna.
For each transfection sample, prepare in separate 1. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. With only a fiveminute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Transfection efficiencies lower than 50% were not reported in this table. Make sure that cells are greater than 90% confluent and vary dna. Invitrogen lipofectamine 2000 transfection reagent 1. It is best to use polypropylene instead of polystyrene tubes. In animal cells, transfection is the preferred term as.
Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Superior performance for cotransfection of sirna and plasmid dna. The studies above reveal that the tri and di peptide lipids have different efficiency for dna and sirna transfection in vitro. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations. The method illustrated is the gelatindna method of the reverse transfection approach. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. To date, various techniques have been used to deliver dna into mammalian cells, including viral vectors, nanoparticles, and transfection agents tas. Transfection and genome engineering fisher scientific. Add the dnalipid complexes dropwise to the culture media.
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. Replace transfection media with culture medium after 3 h. Original article an improved method for increasing the efficiency of. Invitrogen have added a new product, lipofectamine ltx, to their extensive range of transfection products. The intracellular trafficking mechanism of lipofectamine. The transfection with enhanced green fluorescent protein egfpencoding dna was carried out both with nanoparticles and with lipofectamine and followed by. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Dna transfection is the process by which dna is taken up by eukaryotic cells. Following on from other products in the lipofectamine range, lipofectamine ltx is a lipid based reagent for transfecting dna and vector based rnai into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred. Transfection guide overview of transfection methods. Co transfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Viafect transfection reagent transfection assay e4981.
Finally, we present a fast and efficient method for transfection of trypsinized mes cells using the liposomalbased lipofectamine 2000. Lipidmediated dna and sirna transfection efficiency. Im trying to ascertain the volume of the transfection mixture in which the cells are incubated for transfection to. The ability to introduce dna into cultured cells has provided a powerful means to study the function and control of mammalian genes. Due to their high te for both dna and sirnamirna across a broad range of cell lines, including difficulttotransfect cells, they are considered. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Mammalian cell penetration, sirna transfection, and dna. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and mrna into a wide. Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene.
Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. 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 and suspension cell lines. It consistently produces high transfection efficiency and high protein overexpression. Realizing their full potential, however, requires nucleic acid delivery reagents that are simple to prepare, effective across many mammalian cell lines, and nontoxic. Plate cells so they will be 7090% confluent at the time of transfection. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. However, in comparing the transfection efficiency of the two cationic lipids, lipofectamine ltx seems to perform better than lipofectamine 2000 at low dna amounts, while lipofectamine 2000 outperforms lipofectamine ltx at higher dna amounts. Although dna and sirna are both nucleic acids and have anionic phosphodiester backbones with identical negative charge. The intracellular trafficking mechanism of lipofectaminebased. Lipofectamine 2000 transfection protocol 10cm plates reagents.
An electroporation protocol for efficient dna transfection. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. The transfected cell microarray forms in 12 days and is then ready for downstream assays. Nucleic acid reagents, including small interfering rna sirna and plasmid dna, are important tools for the study of mammalian cells and are promising starting points for the development of new therapeutic agents. Typically, dna is suspended in sterile water or te buffer to a final concentration of 0. I use 24well plates for transfections in triplicates. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. While the application of bacterial transformation is typically for overexpression of genes in an effort to produce large quantities of proteins that can be purified and studied, dna transfection is typically used for the studying the effect of gene expression in a. When plasmids encoding fluorescent proteins are transfected, check transfection efficiency 24 h posttransfection with an inverted fluorescence microscope zeiss axiovert a1.
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