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. Lipofectamine 2000 thermo fisher scientific and 25 nm sirna according to each manufacturers protocol. Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Lipofectamine 2000 and lipofectamine rnaimax transfection. 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. Multiplexed crisprcas9 gene knockout with simple crrna. 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. For additional information on scaling your transfection reaction, see page 4. Consequently, we examined the ability of a commercial pei preparation to allow the introduction of foreign genes into.
Invitrogen lipofectamine 2000 transfection reagent. Cotransfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. Cell culture, transfection and coimmunoprecipitation assay cos7 cells were maintained in dulbelccos modified eagles medium supplemented with 10% volvol fetal bovine serum invitrogen. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Xtremegene 9 dna transfection reagent polymer reagent for. For example, for cotransfection of plasmid dna and sirna in a 24well plate, we would recommend using. D the transfection of n2a1 into mouse c10 cells using lipofectamine 2000 resulted in rfppositive cells by 72 h. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 2000 is useful for transfecting dna plasmid into hek293 cells 4.
One is pcdna3 based protein a and the other is pcdna5frtto based gfptagged protein b. Lipofectamine 2000 right panel around 5% efficiency. For other tissue culture formats, refer to the user protocol pdf. This procedure can be modified for alternative packaging cell lines or transfection reagents. Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent. 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. Invitrogen lipofectamine 2000 transfection reagent fisher. 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. Cotransfection of two plasmids tissue and cell culture. Recent data, however, suggest that linear polymers of the cation polyethyleneimine pei can facilitate the uptake of nucleic acids by neurons. Invitrogen lipofectamine 2000 transfection reagent 0. Transfection is the process of inserting genetic material, such as dna and double stranded rna, into mammalian cells. A sample protocol is listed here for transfection experiments performed in 6well plates. This protocol can be used when using other dna transfection reagents such as transit2020.
Rnai, sirna, shrna plasmid or mirna plasmid into mammalian cells using lipofectamine 2000 reagent are described in this section. Lipofectamine 2000 dna transfection reagent protocol. Xtremegene 9 dna transfection reagent is a nonliposomal multicomponent reagent for. 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. Parameters influencing highefficiency transfection of. A guide to transient expression of membrane proteins in hek.
This protocol can be used to produce lentivirus from a lentiviral vector transfected into lentix 293t cells using a polyethyenimine pei transfection protocol. Cotransfection of 2 plasmids in hek293 cells i am trying to cotransfect 2 plasmids in hek293 cells. This protocol can also be followed when cotransfecting plasmid dna and mirna by simply. 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. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. Co transfection of 2 plasmids in hek293 cells i am trying to co transfect 2 plasmids in hek293 cells. 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. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine 2000 transfection protocol 10cm plates reagents.
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. 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. 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. The table below lists the range of recommended reagent amounts and volumes to use to transfect cells in various tissue culture formats. Transfection efficiencies lower than 50% were not reported in this table. We did not find substantial transfection efficiency variations in different batches of lipofectamine 2000. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. 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.
For other tissue culture formats, refer to the user protocol. We did not find substantial transfection efficiency variations in. Cotransfection protocol for plasmid dna and sirnamirna. This protocol can also be followed when cotransfecting plasmid dna and mirna by simply substituting mirna for sirna. 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. 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. Lipofectamine 2000 transfection reagent from invitrogen. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Transfection efficiencies were optimal around 40% of transfected cells per spot with 11.
Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rna molecules. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Cotransfection protocol for multiple plasmid dnas mirus bio. Plate cells so they will be 6080% confluent at the time of transfection. Lipofectamine 3000 and rnaimax outperformed lipofectamine 2000 in hek 293 cells figure s1. Amounts of dna and reagent jetprime and competitors per well in 6well plate for transfection according to manufacturers recommendations. In cotransfection experiments optimized for cell viability, dharmafect duo reagent resulted in both reliable plasmid expression and potent sirnamediated knockdown. 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 is a proprietary blend of lipids and other components supplied in 80% ethanol, filtered through 0. C using et cloning 6, a cmvrfp expression cassette was introduced into the pbelobac11 vector of 265a8 to generate bac n2a1. Lipofectamine 2000 transfection reagent is the number one choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna.
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. Transfection guide overview of transfection methods promega. 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. Rapid and highly efficient mammalian cell engineering via. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. 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. 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. Usa 800 424 6723 australia 61 2 9914 2800 austria 01 877 89 01 belgium 09 385 55 11 brazil 55 31 3689 6600. 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. 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. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng final lipofectamine 2000 reagent per well 0. A comparison showing transfection efficiency of polyjet reagent vs.
In co transfection experiments optimized for cell viability, dharmafect duo reagent resulted in both reliable plasmid expression and potent sirnamediated knockdown. 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. Genscript recommends using lipofectamine 2000 for all transfections. Outlined below is an easytofollow cotransfection protocol for plasmid dna and sirna using transitx2 dynamic delivery system in a 6well format. As always, use good cell culture practices and wear your personal protective equipment. Highly recommended reagent for those who intend to use co transfection.
Polyethyleneiminemediated transfection of cultured. Lipofectamine 2000 reagent thermo fisher scientific. Another advantage of lipofectamine 2000 is its application for cotransfection experiments in which plasmid dna and sirna are used simultaneously. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. The protocol presented here describes the synthesis of a dendronized polymer as a highly efficient and nontoxic transfection agent. Life science group bulletin 5969 rev a useg 100503 0310 sig 1109 biorad laboratories, inc.
To transfect cells with sirna, follow the protocol as described for dna but. Guidelines for cotransfection of adherent cells with sirna. Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. The complexes are prepared inside the wells, after which cells and medium are added. 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.
L3000001 lipofectamine 3000 transfection reagent sparshbio. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. This protocol is based on that from life technologies called. 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. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Co transfection of multiple plasmid dnas is a technique increasingly employed by biology researchers. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. 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.
Despite this, a satisfactory mechanismbased explanation of their. Transient transfection was performed with lipofectamine 2000 according to the manufacturers protocol invitrogen. For uptodate licensing information and productspecific disclaimers, see the respective qiagen kit handbook or user manual. Sirna and plasmid cotransfection using lipofectamine 3000. Once produced, lentivirus can be used for a variety of downstream applications such as.
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