Lipofectmax transfection reagent has been tested to work the same efficiency as lipofectamine 2000 reagent, and used for the transfection of both. Effects of lipofectamine 2000sirna complexes on autophagy. Lipofectamine 2000 transfection reagent life technologies, cat. Transfection protocol lipofectamine 3000 transfection. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Versatileone reagent for dna, rna, and cotransfection efficiently transfect difficulttotransfect cells lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. In contrast, transfection of cells with lipofectamine 2000mrna complexes in optimem i reduced serum media or the use of transit mrna transfection reagentmrna complexes in cell culture. A sample protocol is listed here for transfection experiments performed in 6well plates. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Lipofectamine 2000 is commonly used for sirna transfections. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Lipofectamine 2000 is a cationlipid formulation transfection reagent that forms liposomes in aqueous media.
Effects of lipofectamine 2000sirna complexes on autophagy in. Due to their high te for both dna and sirnamirna across a broad range of cell lines, including difficulttotransfect cells, they are considered. 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. Reagents compared to lipofectamine 2000 at 24 hours. The intracellular trafficking mechanism of lipofectaminebased. Lipofectamine 2000 is primarily used for delivery of plasmid dna, but it also works well for delivery of sirna and mirna mimics and of course codelivery. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Genetic transfection an overview sciencedirect topics. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. The purpose of this study is to evaluate the effect of sirna transfection using lipo2000. I use 24well plates for transfections in triplicates. Optimem reduced serum medium, no phenol red life technologies, cat. Lipofectamine 2000sirna complexes cause endoplasmic.
Lipofectamine 2000 and lipofectamine 3000 reagents deliver dna or cotransfect with excellent transfection. Apr 14, 2009 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. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Evaluation of transfection methods for transient gene. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Lipofectamine 2000 transfection reagent is a proprietary formulation. Lipofectamine 2000 reagent thermo fisher scientific.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Certain chemical reagents such as calcium phosphate and lipids bind to dna or rna and deliver transfection complex via. For a procedure to transfect mammalian cells with short interfering rnas sirna for use in rna interference. Plate cells so they will be 6080% confluent at the time of transfection. Mammalian cell penetration, sirna transfection, and dna transfection by supercharged proteins brian r. A b 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. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the.
Mar 03, 2014 the transfection of ecs with stemfect rna transfection reagent and lipofectamine 2000 in cell culture medium resulted in very low levels of transfection. Optimized conditions for successful transfection of human. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Arpe19 transfection kit retinal pigment epithelium. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. Add 6l of lipofectamine2000 from biostores and 100 l of. Cells in a 6 well dish, plated and at 70% confluence.
Advanced transfection with lipofectamine 2000 reagent. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000, 3000, messengermax, and rnaimax. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. Jun 10, 2011 lipofectamine 2000 is commonly used for sirna transfections. See more supporting information at this feature is not available right now. Small interfering rna sirna highthroughput screening has a variety of.
Lipofectamine 2000, 3000, messengermax, and rnaimax reagents are cationiclipid transfection reagents formulated for superior transfection efficiencies on a variety of cell types. The newer transfection reagents lipofectamine 2000 dalby et al. 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. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent.
Transfection protocol transfection components component cat. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. Transfecting sirna using lipofectamine rnaimax pin. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Can i use the lipefectamine2000 for mimic mirna transfection. Invitrogen lipofectamine 3000 transfection reagent. As always, use good cell culture practices and wear your personal protective equipment. Liu1 howard hughes medical institute, department of chemistry and chemical biology, harvard university, 12 oxford street, cambridge, ma 028. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy.
To transfect cells with sirna, follow the protocol as. Stealthsirna transfection protocol lipofectamine 2000. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Lipofectamine 2000 lipo2000 delivery system is commonly used for short interfering rna sirna transfection, whereas the cellular responses have attracted little attention. 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. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and.
Serumfree medium is necessary for successful dnalipofectamine 2000. Highest transfection efficiency in many cell types and formats e. Add 1 ul lipofectamine 2000 into 30 ul dmem without serum, mixing well and wait for 5 min at room temperature. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Lipofectamine 2000, 3000, messengermax, and rnaimax transfection reagents green benefits sustainable packagingno dry or gel ice, no expanded polystyrene eps coolers introduction we are committed to designing our products with the environment in mindits part of how we enable our customers to make the world healthier, cleaner, and. 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 protocol lipofectamine 3000 transfection reagent gct. Rna transfection is the process of deliberately introducing rna into a living cell. 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. Transfection of messenger rna mrna is a promising alternative to pdna or viral vector to achieve target protein expression, particularly in nonproliferative cells such as primary human cells 19,20. Transfection of messenger rna mrna is a promising alternative to pdna or viral vector to achieve target protein expression, particularly in nonproliferative cells such as. The trusted, simple, broadspectrum transfection reagent for most cell lines.
Detailed transfection procedures are also available. We did not find substantial transfection efficiency variations in. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. For each transfection sample, prepare in separate 1. Lipofectamine 2000 transfection reagent dartmouth college. The impact of transfection mediated toxicity mirus bio. Optimization of sirna transfection for breast and glioma. Generally speaking, transfection is a tool which may be used to introduce charged dna and rna molecules into cells, which have a negatively charged membrane. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cellculture treated multidishes 142475 step tube complexation components amount per well 24well. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. However, few studies have examined cellular responses to this delivery system. Dna is enclosed within the liposomes and the complexes fuse with the host cell membrane for dna entry into the cytoplasm and eventu ally into the nucleus.
Treatment with lipofectamine 2000 and cy3sirna resulted in. 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. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine 2000 and lipofectamine rnaimax transfection. Invitrogen lipofectamine 2000 transfection reagent 1. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Optimization of sirna transfection for breast and glioma cancer cell cultures rna silencing technology and methods based thereupon are important in todays medical biotechnology. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Hela cells were transfected with a gfp encoding plasmid dna using either transitlt1, transit.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Rna can be purified from cells after lysis or synthesized from free nucleotides either chemically, or enzymatically using an rna polymerase to transcribe a dna template. For additional information on scaling your transfection reaction, see page 4. 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. Lipofectamine transfection of c2c12 cells bridges lab. As with dna, rna can be delivered to cells by a variety of means including microinjection, electroporation, and lipidmediated transfection. For transfection of cells only with optimem i reduced serum media, the lipofectamine 2000 transfection mixtures were prepared by addition of 2. Mammalian cell penetration, sirna transfection, and dna. Prewarm 50ml of optimum stored in cold room at 4c 10min. Plate cells so they will be 7090% confluent at the time of transfection. 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 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Lipofectamine rnaimax has a broad peak of activity. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Invitrogen lipofectamine 2000 transfection reagent life. 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. 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 of nih3t3 cells, hela, swis 3t3, 293t with. Do not add antibiotics to media during transfection as this causes cell death.
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