Our lipofectamine reagentspecific protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types see download protocol links in the table below. Lipofectamine 3000 reagent thermo fisher scientific id. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Each reagent was tested with a range of transfection reagent.
Lipofectamine rnaimax rnaimax, a new cationic lipofection reagent, has shown higher efficiency in delivering mirna into many stem cells and cell lines zhao et al. The intracellular trafficking mechanism of lipofectaminebased. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to transfect 17 cell lines with a gfpexpressing plasmid in a 24well plate format, using 0. Highly efficient transfection of a human epithelial cell line with chemically synthesized sirna using silentfect lipid reagent, rev a.
Using a systematic approach, we screened 60 transfection reagents using six commonlyused mammalian cell lines and identified a novel. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types. Lipofectamine 2000 reagent thermo fisher scientific. Xtremegene hp dna transfection reagent sigmaaldrich. The intracellular trafficking mechanism of lipofectamine. Increased transfection efficiency with the freezethawed reagent was also seen with. Culture plates were incubated at 37c and 5% co2 in shaking at 120 rpm.
The commercial transfection reagent lipofectamine has been widely used for cytoplasmic delivery of nucleic acids and for cytosolic engagement with intracellular innate immune sensors to trigger type i interferon ifn production. Lipofectamine transfection reagent is one of our first generation cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. A new transfection reagent for effective transfection of. It is widely accepted that the final transfection efficiency te of lipidbased transfection reagents trs is ratelimited by several biological barriers such as cellular uptake, intracellular trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8. Designed to transfect a broad range of eukaryotic cells, including insect cells, many cell lines not transfected well by other reagents, and hardtotransfect cell lines. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. As a matter of fact, they should be evaluated in their entirety for the development of optimized nonviral gene delivery vectors.
Make dna lipofectamine 3000 complexes in serumfree medium such as. Lipofectaminea rnaimax reagent thermo fisher scientific. Invitrogen lipofectamine 2000 transfection reagent 1. Lipofectamine 3000 reagent is suitable for novel genome editing applications.
Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Plasmids were transfected using lipofectamine ltx reagent with plus reagent in optimem according to the manufacturers instructions. Lipofectamine 2000 dna transfection reagent protocol. Effect of cell density on total reporter gene activity. Lipofectamine crisprmax transfection reagent is a proprietary formulation for transfecting cas9 nucleasegrna complex into a wide range of eukaryotic cells. Lipofectamine 3000 reagentefficient, reproducible transfection for biologically relevant cell models introduction with the increase of research in more biologically relevant cell models, existing molecular and cellular techniques need to be improved. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 3000 reagent thermo fisher scientific us. May 11, 2016 it is widely accepted that the final transfection efficiency te of lipidbased transfection reagents trs is ratelimited by several biological barriers such as cellular uptake, intracellular trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8. There are several ways in which to introduce cas9guide rna complexes into cells. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. Lipofectamine ltx reagent with plus reagent from thermo. Lipofectamine 2000 is a highperformance transfection reagent for gene expression and gene silencing.
Dna lipofectamine complexes must be made in serumfree medium such as optimem reduced serum medium and can be added directly to cells in culture medium, in the presence or absence of serumantibiotic. Transfection of caco2 cells with sirna using the silentfect lipid reagent, rev a. Lipofectamine o23, ot, hela, 2a1 wt shsy5y use 10x amount of dna polyfect 292t, hela, mcf7, ot70, otbag references. The amount of lipofectamine 2000 should be changed in proportion to the culture medium. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Invitrogen lipofectamine 3000 transfection reagent. Prepare 6 well plates by adding bakedsterile 22x22 1.
Lipofectamine ltx reagent is an advanced solution for gene expression in both common and challenging cell types. It increases the likelihood of successful cleavage and recombination with talens or crisprs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes. Lipofectamine reagents go online to view related products. A new transfection reagent for effective transfection.
Transfection reagent lipofectamine triggers type i. Examples of lipofection transfection reagents may include dotma n12,3,dioleyloxypropyln,n,ntrimethylammonium. Plasmid dna, synthetic sirna, rnai plasmids shrna, mir content and storage. Lipofectamine transient transfection of cos7 cells to. Mia paca2 transfection kit pancreatic carcinoma altogen. Lipofectamine transfection reagent thermo fisher scientific.
High efficiency gene transfer by multiple transfection. Transfection of cas9 rnp ribonucleoprotein into adherent. Lipofectamine 2000 transfection reagent dartmouth college. Briefly vortex the xtremegene 9 dna transfection reagent vial. Lipofectamine ltx reagent with plus reagent from thermo fisher scientific description lipofectamine ltx reagent is a plasmid transfection reagent that offers the right balance of potency and gentleness for your cells, resulting in the highest possible transfection efficiencies and viabilities. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. Improve lentiviral production using lipofectamine 3000 reagent.
Works effectively with all common cell lines as well as with many challenging ones, and can. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine transfection reagent from thermo fisher. Effectene transfection reagent handbook qiagen lipofectamine transfection reagent handbook invitrogen polyfect transfection reagent handbook qiagen. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Researcher quotes see full list ordering information gentle with low toxicity. Lipofectamine ltx reagent is a plasmid transfection reagent that offers the right balance of potency and gentleness for your cells, resulting in the highest possible transfection efficiencies and viabilities. Gfp expression was analyzed 48 hours posttransfection. This study shows that lipofectamine 2000 is an efficient reagent for the delivery of. Lipofectamine 2000 transfection reagent from thermo fisher.
Dilute lipofectamine rnaimax reagent in optimem medium 2 dilute sirna in optimem medium 3 add diluted sirna to diluted lipofectamine rnaimax reagent 1. Lipofectamine reagents offer you the ability to consistently transfect many cell types with high efficiency and exceptional ease of use. Dnareagent complexwell 10 l 50 l 250 l incubate cells for days at 37c amount 96well 24well 6well dnawell 100 ng 500 ng 2500 ng lipofectamine 2000 reagent well 0. To get a better understanding of how each reagent performs on a variety of cell lines, a transfection trial was performed with the three promega reagents and the leading competitors transfection reagent. A high efficiency transfection protocol employing a common polycationic lipid is described. Our lipofectamine reagent specific protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types see download protocol links in the table below. Dnalipofectamine complexes must be made in serumfree medium such as optimem reduced serum medium and can be added directly to cells in culture medium, in the presence or absence of serumantibiotic. Whats the difference between lipofectine and lipofectamine. Dilute xtremegene 9 dna transfection reagent with serumfree medium, such as optimem to a concentration of 3. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Despite this, a satisfactory mechanismbased explanation of their.
The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. Lipofectamine 3000 protocol written out with timing. Lipofectamine rnaimax transfection reagent from thermo. At this moment, i am only able to obtain 510% transfection efficiency. Invitrogen lipofectamine ltx reagent with plus reagent 0. Refer to the cell lines database for a list of cell types successfully transfected. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results.
In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over 50,000 references to date. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. The advantages of lipidbased transfection reagents have permitted their widespread use in molecular. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas e. The optimal amount of dna to use in the transfection will vary widely depending upon the type of dna, transfection reagentmethod, target cell line, and number of cells genetic material dna quality and quantity factors affecting transfection 8. Using lipofectamine, a widely used transfection reagent, we transfected 293t cells with a plasmid harboring the. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Improved delivery of cas9 proteingrna complexes using. Lipofectamine ltx reagent is a plasmid transfection reagent that offers a balance of potency and gentleness for your cells, resulting in high transfection efficiencies and viabilities. Transitlt1 transfection reagent research reagent products. Transitlt1 is a low toxicity, serumcompatible transfection reagent that eliminates the need for any culture medium change. Invitrogen lipofectamine ltx reagent with plus reagent.
Advanced transfection with lipofectamine 2000 reagent. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Viafect transfection reagent transfection assay e4981. 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. You can generally use other serumfree medium instead of optimem. Highest transfection efficiency in many cell types and formats e. Pdf optimization of sw480 colon cancer cells transfection. Cas9 nuclease may be used in vivo to create targeted genome modifications. Invitrogen lipofectamine 3000 transfection reagent 0. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Dna reagent complexwell 10 l 50 l 250 l incubate cells for days at 37c amount 96well 24well 6well dnawell 100 ng 500 ng 2500 ng lipofectamine 2000 reagent well 0. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Cho transfection kit chinese hamster ovary cells altogen. Lipofectamine 3000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to transfect cells. Lipofectamine rnaimax transfection reagent from thermo fisher. Invitrogen lipofectamine 2000 transfection reagent life. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Optimization of sw480 colon cancer cells transfection with lipofectamine 2000.
Lipidbased transfection reagents exhibit cryoinduced increase in. Invitrogen lipofectamine 3000 transfection reagent fisher. Invitrogen lipofectamine 2000 transfection reagent 0. When developing lipofectamine 3000 reagent, we optimized all four steps in the transfection process and combined our most advanced lipid nanoparticle technologies together. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. This reagent provides an advanced solution for gene expression in common cell lines, particularly chinese hampster ovary cho, and challenging cell types such. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid.
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