The intracellular trafficking mechanism of lipofectamine. Incubate for 20 min at rt 6 after incubation, add optidnalipo mixture to cells dropwise. Livecell imaging to compare the transfection and gene. Lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. We did not find substantial transfection efficiency variations in. Plate cells so they will be 7090% confluent at the time of transfection. The intracellular trafficking mechanism of lipofectaminebased. An electroporation protocol for efficient dna transfection. Im trying to ascertain the volume of the transfection mixture in which the cells are incubated for transfection to. 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. The transfected cell microarray forms in 12 days and is then ready for downstream assays. Genscript recommends using lipofectamine 2000 for all transfections. With only a fiveminute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability.
Finally, we present a fast and efficient method for transfection of trypsinized mes cells using the liposomalbased lipofectamine 2000. 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. Highest transfection efficiency in many cell types and formats e. Transfection efficiencies lower than 50% were not reported in this table. 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.
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. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations. Liposome lipidbased formulation has the least influence. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transfection guide overview of transfection methods.
Due to their high te for both dna and sirnamirna across a broad range of cell lines, including difficulttotransfect cells, they are considered. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Invitrogen lipofectamine 2000 transfection reagent 1. For each transfection sample, prepare complexes as follows. Lipofectamine 2000 transfection reagent dartmouth college. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. Mammalian cell penetration, sirna transfection, and dna. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. In animal cells, transfection is the preferred term as. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Product isguaranteed for 6 months from the date of shipment if stored properly.
Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Add 250 ul optilipo 2000 mixture to each tube with dna. Although dna and sirna are both nucleic acids and have anionic phosphodiester backbones with identical negative charge. Descriptionlipofectamine 2000 is a proprietary formulation suitable for the transfection of. 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. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Realizing their full potential, however, requires nucleic acid delivery reagents that are simple to prepare, effective across many mammalian cell lines, and nontoxic. Typically, dna is suspended in sterile water or te buffer to a final concentration of 0. Lipofectamine 2000 dna transfection reagent protocol. Transfection and genome engineering fisher scientific. Lipofectamine 2000 and lipofectamine rnaimax transfection. Make sure that cells are greater than 90% confluent and vary dna. Original article an improved method for increasing the efficiency of.
Transfection of mcf7 with lipofectamine 2000 maartje vogel. Researchers use lipofectamine 2000 reagent for plasmid dna transfection as well as sirna and shrnabased gene knockdown. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. 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. Replace transfection media with culture medium after 3 h. For each plasmid to be transfected, prepare a separate, sterile 1. 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. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and mrna into a wide. Prepare plasmid dnalipid complexes recommend 2 doses of lipid.
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. Lipofectamine 2000 transfection protocol 10cm plates reagents. Superior performance for cotransfection of sirna and plasmid dna. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. After removal of the transfection reagent, the slide is placed in a culture dish and covered with cells in media. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. For each transfection sample, prepare in separate 1. 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. Fast and efficient transfection of mouse embryonic stem. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfection protocol for hpc7 using lipofectamine 2000 igem 2017.
The ability to introduce dna into cultured cells has provided a powerful means to study the function and control of mammalian genes. To date, various techniques have been used to deliver dna into mammalian cells, including viral vectors, nanoparticles, and transfection agents tas. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Viafect transfection reagent transfection assay e4981. Lipofectamine stem reagent was designed specifically for stem cells and confers low cytotoxicity, enabling researchers to achieve optimal. Add the dnalipid complexes dropwise to the culture media. As expected, transfection efficiency for both reagents correlates with the amounts of dna used. Dna transfection is the process by which dna is taken up by eukaryotic cells. The method illustrated is the gelatindna method of the reverse transfection approach. Lipidmediated dna and sirna transfection efficiency.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. The studies above reveal that the tri and di peptide lipids have different efficiency for dna and sirna transfection in vitro. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. 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. It is best to use polypropylene instead of polystyrene tubes. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate. This commercially available transfection agent consists of positively charged lipid molecules. 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. Dnaintm transfection reagent family a comparative study of transfection reagents. It consistently produces high transfection efficiency and high protein overexpression. When plasmids encoding fluorescent proteins are transfected, check transfection efficiency 24 h posttransfection with an inverted fluorescence microscope zeiss axiovert a1. Despite this, a satisfactory mechanismbased explanation of their.
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