A new transfection reagent for effective transfection of. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. You can generally use other serumfree medium instead of optimem. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. The optimal level of each transfection reagent was determined empirically and all reagents were used according to manufacturers protocol. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. The reagent provides high transfection efficiency in many cells types and formats. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature.
The protocal is from thermo fisher scientific important guidelines for transfection 1. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Lipofectamine ltx reagent with plus reagent protocol optimized for efficiency. Lipofectamine 2000 transfection reagent from invitrogen. Delivery of episomal vectors into primary cells by means of. The main objective of this study was to optimize transfection of sw480 colon cancer cells with lipofectamine 2000. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. 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.
Lipofectamine 2000 from invitrogen biocompare product. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic. Highest transfection efficiency in many cell types and formats e. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Lipofectamine 3000 reagent thermo fisher scientific fr. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
Numerous chemical and physical approaches have been used to deliver the dna, but their efficacy has been variable and is highly dependent on the cell type to be transfected. 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. The intracellular trafficking mechanism of lipofectamine. Superior performance for cotransfection of sirna and plasmid dna.
Invitrogen lipofectamine 2000 transfection reagent fisher. This observation is consistent with reports that, regardless of the transfection protocol, delivery of. The average lentiviral titers in our preparations are around 5 x 10. On measuring mirnas after transient transfection of mimics or. The average lentiviral titers in our preparations are around 56x 10 5 x 10. Invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. The ratio of transfection reagent to episomal vectors was varied, and the subsequent. It is used to increase the transfection efficiency of rna including mrna and. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Pool dishes of cells that have been prepared for transfection. The mechanism of lipofectamine 2000 mediated transmembrane.
Invitrogen lipofectamine 3000 transfection reagent from. Lipofectamine 2000 transfection reagent, invitrogen. Scaling up or down lipofectamine 3000 reagent transfections. Invitrogen lipofectamine 2000 transfection reagent 0. Evaluation of transfection methods for transient gene. Lipofectamine ltx reagent offers a new, advanced solution for gene expression. Transfections were performed in 24well plates using 0. 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. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. Studies were undertaken to evaluate and compare the transfection efficacy. Lipofectamine ltx reagent is a plasmid transfection reagent that offers a balance of potency and gentleness for your.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. Researchers use lipofectamine 2000 reagent for plasmid dna transfection as well as sirna and shrnabased gene knockdown. 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.
Lipofectamine 2000 procedure for simultaneously transfecting. Liposomebased dna carriers may induce cellular stress. Photograph of expected results dna lipofectamine scaling up or down. Although invitrogen recommends their own brand of culture medium for transfection, called optimem, we found no difference between this and conventional media. 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. Superior efficiency into the broadest spectrum of difficulttotransfect cell types. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. Plate cells so they will be 7090% confluent at the time of transfection.
Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. On measuring mirnas after transient transfection of mimics. Dilute lipofectamine 3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. A new transfection reagent for effective transfection of primary cells, hardtotransfect cells and sensitive established cell lines.
Optimem reduced serum medium, no phenol red life technologies, cat. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. 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. Optimized conditions for successful transfection of human. This work was aimed to understand better the mechanism of this phenomenon and its potential. Lipofectamine 2000 transfection protocol 10cm plates reagents. During functional studies on the rat stressinducible hspa1b hsp70. Invitrogen lipofectamine 3000 transfection reagent. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. The intracellular trafficking mechanism of lipofectaminebased. Outline of protocol steps for the use of lipofectamine 2000 reagent. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. The purpose of this protocol is to introduce dna or small rna into eukaryotic cells for recombinant expression or down regulation of defined.
The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for 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. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells. The intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Cells were harvested with trizol reagent invitrogen. Prewarm 50ml of optimum stored in cold room at 4c 10min. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. 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. Lipofectamine 3000 reagent outperforms invitrogen lipofectamine 2000 and fugene hd reagents.
Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Invitrogen lipofectamine 3000 transfection reagent fisher. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. This study shows that lipofectamine 2000 is an efficient reagent for the delivery of genes into sw480 cells. In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. Add 6l of lipofectamine2000 from biostores and 100 l of. Versatileone reagent for dna, rna, and cotransfection. Altogen biosystems provides efficient biodegradable transfection reagent kits for the caco2 colon cancer cell line. 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. Pdf optimization of sw480 colon cancer cells transfection with.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. 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. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1.
Invitrogen lipofectamine 3000 transfection reagent 1. Strategy used in this study and results of a methylationbased analysis of mirnas in a panel of hcc cell lines and control liver tissues. Pdf primary cells and certain established cells have generally been refractory to transfection using liposomesbased methods. The use of these different lipofectamine 2000 amounts for transfection showed no significant differences. Jun 10, 2011 during functional studies on the rat stressinducible hspa1b hsp70. The neuronal cytoskeleton, motor proteins, and organelle trafficking in the axon. As a matter of fact, they should be evaluated in their entirety for the development of optimized nonviral gene delivery vectors. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. The delivery of dna into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Lipofectamine 2000 reagent thermo fisher scientific. Amongst the eleven transfection reagents investigated, lipofectamine 2000, lipofectamine rnaimax and dharmafect 3 consistently gave the best results, in terms of sirna uptake and target gene knockdown with minimal cell toxicity and type i ifn response induction. Lipofectamine ltx with plus reagent thermo fisher scientific fr. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine transfection reagents offer efficient delivery of dna, rna, or sirnamirna.
Enable high transfection efficiency in novel genome editing applications. I use 24well plates for transfections in triplicates. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. 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. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Plate cells so they will be 70 90% confluent at the time of transfection. Pdf optimization of sw480 colon cancer cells transfection.
Simple protocol and efficient transfection of primary cells and macrophages. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and. Lipofectamine 2000 was simple to use and unlike other reagents displayed very low toxicity. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
For each of the cell lines tested, there was less than 5% difference in the. Advanced transfection with lipofectamine 2000 reagent. The diluted dna and lipofectamine 2000 were combined and incubated at. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Invitrogen lipofectamine 2000 transfection reagent. Optimem 1 bottle life technologies 31985070 lipofectamine 2000 1ml vial life technologies 11668019. The trusted, simple, broadspectrum transfection reagent for most cell lines.
Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 is then added to culture medium within a sterile tube along with the dna at a 3. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown. This study shows that lipofectamine 2000 is an efficient reagent. Lipofectamine 3000 protocol written out with timing. In addition, invitrogen claims that lipofectamine 2000 gives the most proven results in more functional and phenotypic assays than any other reagent and they back up this claim with an abundance of references. Lipofectamine 2000 to use for a549 transfection a volume of 2 ul of lipofectamine 2000 do you think will be enough for an overnight transfection in dmemno pens strep 100 nm sirna. The inhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000 pgfpn2.
Ambion into hcc cell lines using lipofectamine 2000 invitrogen. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. A combination of experiments in silico, molecular analyses in human hcc cell lines and the analysis of primary human hcc specimens with corresponding noncancerous liver tissues and normal liver was. Comparison of small interfering rna sirna delivery into. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and. Dec 01, 2006 in addition, invitrogen claims that lipofectamine 2000 gives the most proven results in more functional and phenotypic assays than any other reagent and they back up this claim with an abundance of references.
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