Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Sirna and plasmid cotransfection using lipofectamine 3000. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Plate cells so they will be 6080% confluent at the time of transfection. Outlined below is an easytofollow cotransfection protocol for plasmid dna and sirna using transitx2 dynamic delivery system in a 6well format. Guidelines for cotransfection of adherent cells with sirna. 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. For uptodate licensing information and productspecific disclaimers, see the respective qiagen kit handbook or user manual.
Invitrogen lipofectamine 2000 transfection reagent. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. The reagent provides high transfection efficiency in many cells types and formats. Learn more about lipofectamine 2000 cotransfection protocol with lipofectamine 2000 this reference provides general guidelines and a procedure to cotransfect plasmid dna and an rnai molecule i. Rapid and highly efficient mammalian cell engineering via. Lipofectamine 2000 transfection reagent is a superior choice for co transfection, given its effectiveness for transfecting both sirna and plasmid dna ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems. Cotransfection of sirna or mirna reagents with a plasmid reporter system is increasingly common, yet few transfection reagents on the market will effectively deliver both molecules. This protocol can be used when using other dna transfection reagents such as transit2020.
One is pcdna3 based protein a and the other is pcdna5frtto based gfptagged protein b. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Cotransfection of two plasmids tissue and cell culture. Lipofectamine 2000 reagent thermo fisher scientific.
A guide to transient expression of membrane proteins in hek. According to the manufacturer, this step is not necessary due to low cytotoxicity of the reagent, which surely makes the use of lipofectamine 2000 even more convenient. Consequently, we examined the ability of a commercial pei preparation to allow the introduction of foreign genes into. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. For additional information on scaling your transfection reaction, see page 4. Co transfection of 2 plasmids in hek293 cells i am trying to co transfect 2 plasmids in hek293 cells. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems.
Invitrogen lipofectamine 2000 transfection reagent 0. Furthermore, the optimization of the polymer as a co transfection reagent for large and multiple plasmids in cell lines is described, in addition to general considerations for co transfection experiments. Lipofectamine 2000 transfection protocol 10cm plates reagents. Xtremegene 9 dna transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, filtered through 0. Transfection guide overview of transfection methods promega. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. A comparison showing transfection efficiency of polyjet reagent vs. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. May 20, 2019 currently, multiplexed gene cotargeting is a cumbersome process with crisprcas9 that either use the cotransfection of in vitro transcribed cas9 mrna and sgrnas, cotransfection of a mixture of several cas9 rnps, cotransfection of a cas9 vector with multiple sgrna expressing vectors, and transfection or transduction using specially. Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rna molecules.
This protocol can be used to produce lentivirus from a lentiviral vector transfected into lentix 293t cells using a polyethyenimine pei transfection protocol. Lipofectamine 2000 transfection reagent from invitrogen. We did not find substantial transfection efficiency variations in. Usa 800 424 6723 australia 61 2 9914 2800 austria 01 877 89 01 belgium 09 385 55 11 brazil 55 31 3689 6600. This protocol can also be followed when cotransfecting plasmid dna and mirna by simply substituting mirna for sirna. Furthermore, the optimization of the polymer as a cotransfection reagent for large and multiple plasmids in cell lines is described, in addition to general considerations for cotransfection experiments.
Lipofectamine 2000 dna transfection reagent protocol. Transient transfection was performed with lipofectamine 2000 according to the manufacturers protocol invitrogen. Incubate the cells at 37c in a co2 incubator for 2472 hours until they are. Parameters influencing highefficiency transfection of. For example, for cotransfection of plasmid dna and sirna in a 24well plate, we would recommend using. 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. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. The complexes are prepared inside the wells, after which cells and medium are added. Life science group bulletin 5969 rev a useg 100503 0310 sig 1109 biorad laboratories, inc. Rnai, sirna, shrna plasmid or mirna plasmid into mammalian cells using lipofectamine 2000 reagent are described in this section.
Cotransfection of 2 plasmids in hek293 cells i am trying to cotransfect 2 plasmids in hek293 cells. The table below lists the range of recommended reagent amounts and volumes to use to transfect cells in various tissue culture formats. Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. D the transfection of n2a1 into mouse c10 cells using lipofectamine 2000 resulted in rfppositive cells by 72 h.
A few applications that popularly utilize co transfection are virus production, proteinprotein interaction studies, stable cell line generation, or simple addition of reporter dna constructs to normalize experimental output. Currently, multiplexed gene cotargeting is a cumbersome process with crisprcas9 that either use the cotransfection of in vitro transcribed cas9 mrna and sgrnas, cotransfection of a mixture of several cas9 rnps, cotransfection of a cas9 vector with multiple sgrna expressing vectors, and transfection or transduction using specially. This protocol can also be followed when cotransfecting plasmid dna and mirna by simply. 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. Jun 04, 2008 cell culture, transfection and coimmunoprecipitation assay cos7 cells were maintained in dulbelccos modified eagles medium supplemented with 10% volvol fetal bovine serum invitrogen. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Mar 27, 2007 according to the manufacturer, this step is not necessary due to low cytotoxicity of the reagent, which surely makes the use of lipofectamine 2000 even more convenient. Xtremegene 9 dna transfection reagent polymer reagent for.
Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. This procedure can be modified for alternative packaging cell lines or transfection reagents. Xtremegene 9 dna transfection reagent is a nonliposomal multicomponent reagent for. A sample protocol is listed here for transfection experiments performed in 6well plates. Co transfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. In cotransfection experiments optimized for cell viability, dharmafect duo reagent resulted in both reliable plasmid expression and potent sirnamediated knockdown. To transfect cells with sirna, follow the protocol as described for dna but. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells.
Lipofectamine 2000 is useful for transfecting dna plasmid into hek293 cells 4. Cell culture, transfection and coimmunoprecipitation assay cos7 cells were maintained in dulbelccos modified eagles medium supplemented with 10% volvol fetal bovine serum invitrogen. Cotransfection protocol for plasmid dna and sirnamirna. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. This protocol is based on that from life technologies called. The protocol presented here describes the synthesis of a dendronized polymer as a highly efficient and nontoxic transfection agent. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Invitrogen lipofectamine 2000 transfection reagent strong performance in a wide range of cell lines reliableone of the mostcited dna reagents strong performancefor easytotransfect to moderately difficulttotransfect cell lines easy to usesimple protocol for easy optimization in a wide range of cells. For other tissue culture formats, refer to the user protocol pdf. Amounts of dna and reagent jetprime and competitors per well in 6well plate for transfection according to manufacturers recommendations. 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. Multiplexed crisprcas9 gene knockout with simple crrna. Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent. In co transfection experiments optimized for cell viability, dharmafect duo reagent resulted in both reliable plasmid expression and potent sirnamediated knockdown.
The commonlyused plasmid dna or rna lipofectamine transfection reagent had been shown to be able to deliver many proteins into cells, so we tested several lipofectamine variants for their ability to efficiently deliver cas9 rnps sells et al. Lipofectamine 2000 and lipofectamine rnaimax transfection. Cotransfection of two plasmids need the protocol for lipofectamine 2000 based transfection nov 012009 hi, i am trying to cotransfect transient trasnfection two different plasmids in to hek293t cells. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Reverse transfection protocol reverse transfection combines dna or rna, transfection reagent, and cells in an altered sequence compared to traditional lipofectamine 2000 or magnetofectamine transfection protocols. Invitrogen lipofectamine 2000 transfection reagent fisher. Once produced, lentivirus can be used for a variety of downstream applications such as. Lipofectamine 2000 transfection reagent is easy to usesimply mix with nucleic acid and add to cell culture. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng final lipofectamine 2000 reagent per well 0. Lipofectamine 2000 transfection reagent is the number one choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna. Cotransfection protocol for multiple plasmid dnas mirus bio. Co transfection of sirna or mirna reagents with a plasmid reporter system is increasingly common, yet few transfection reagents on the market will effectively deliver both molecules. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes.
Recent data, however, suggest that linear polymers of the cation polyethyleneimine pei can facilitate the uptake of nucleic acids by neurons. Lipofectamine 2000 thermo fisher scientific and 25 nm sirna according to each manufacturers protocol. Genscript recommends using lipofectamine 2000 for all transfections. For other tissue culture formats, refer to the user protocol. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1.
May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Cotransfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. A few applications that popularly utilize cotransfection are virus production, proteinprotein interaction studies, stable cell line generation, or simple addition of reporter dna constructs to normalize experimental output. 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. Another advantage of lipofectamine 2000 is its application for cotransfection experiments in which plasmid dna and sirna are used simultaneously. Jul 19, 2016 our results suggest that lipofectamine 3000 outperforms lipofectamine 2000 and fugene hd in transfection efficiency andor protein expression, which is consistent with the claims of the manufacturer who reported it for hek293, hela, lncap, hepg2, and a549 cell lines, the superiority of lipofectamine 3000. As a starting point, use an amount of plasmid dna see column 4, dsrna or rnai vector dna see column 5, and lipofectamine 2000 see column 7 that falls around the midpoint of the recommended range, then optimize conditions for your cell line by varying. Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Highly recommended reagent for those who intend to use co transfection.
C using et cloning 6, a cmvrfp expression cassette was introduced into the pbelobac11 vector of 265a8 to generate bac n2a1. Lipofectamine 2000 right panel around 5% efficiency. The intracellular trafficking mechanism of lipofectamine. L3000001 lipofectamine 3000 transfection reagent sparshbio. Transfection efficiencies lower than 50% were not reported in this table. As always, use good cell culture practices and wear your personal protective equipment. Optimization of mouse embryonic stem cell transfection for. Polyethyleneiminemediated transfection of cultured.
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