Combine the two volumes of optimemdna and optimemlipofectamine. 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. 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. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. From the above data in table 3, this indicates that the 1. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Systematic screening of commonly used commercial transfection. I am about to start my stable transfections with lipofectamine 2000. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Add 6l of lipofectamine2000 from biostores and 100 l of. I am getting shitty transfection efficiency using effectene and geneporter 2 transfection reagent. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to. An improved method for increasing the efficiency of gene.
Lipofectamine 2000 dna transfection reagent protocol. Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Help on transfecting mcf7 and hct 116 cells transfection. Plate cells for 212hrs but not over 24 hours prior to.
Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. The transfection protocol for lipofectamine 3000 reagent was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. In tube a place 2 g of dna in optimemi for a final volume of 200 l. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically. Prewarm 50ml of optimum stored in cold room at 4c 10min. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells.
Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. The lenti293t cell line has been tested as a suitable cell line for. Troubleshooting for transfection of the crisprcas9 using lipofectamine. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. As always, use good cell culture practices and wear your personal protective. An example of high efficiency forward and reverse transfection of a549 cells using the broad spectrum transitx2 dynamic delivery system can be found in the figure below. Invitrogen lipofectamine 2000 transfection reagent. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. 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. Invitrogen lipofectamine 3000 transfection reagent from. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. Original article an improved method for increasing the efficiency of. To test this hypothesis, we evaluated the te of lfn and dd on both.
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. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Clinical laboratory products microsatellite instability testing. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Does anyone have experience with the lipofectamine 2000 toxicity.
Lipofectamine 2000 reagent thermo fisher scientific. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338 500 pdf. Invitrogen lipofectamine 2000 transfection reagent 15ml. Examples of cells transfected successfully thermo fisher scientific. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Research diagnostic development diagnostic testing precision medicine preclinical to companion diagnostic development public health.
The impact of transfection mediated toxicity mirus bio. 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. Highest transfection efficiency in many cell types and formats e. Lipofectamine 3000 transfection reagent kit youtube. Viafect transfection reagent transfection assay e4981. What percentage confluency should be used for stable transfection.
Does anyone have experience with the lipofectamine 2000. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Effects of lipofectamine 2000sirna complexes on autophagy in. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. General procedure for transfection of mammalian cells with. I want to try to use hela cell and lipofectamine 2000 to do transfection. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. The manual recommends days whereas some people transfect for a 4. Transient transfection of mammalian cells with lipofectamine.
Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Statistical significance was evaluated by a twotailed students ttest. The trusted, simple, broadspectrum transfection reagent for most cell lines. Lipofectamine transfection of c2c12 cells bridges lab. Enhanced transfection of cryomanipulated lf2000 is not dependent on a specific promoter.
Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Start any new transfection by testing the recommended four. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell. Invitrogen lipofectamine 2000 transfection reagent fisher.
C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. Different cell confluencies, dnareagent ratios and total transfection volumes were optimized for two chemical reagents including jetpei and lipofectamine 2000. I use 24well plates for transfections in triplicates. Invitrogen lipofectamine transfection reagent protocols have been. Studies were undertaken to evaluate and compare the transfection efficacy. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Effects of lipofectamine 2000sirna complexes on autophagy. I want to try to use hela cell and lipofectamine 2000 to. The mechanism of lipofectamine 2000 mediated transmembrane. 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. I have used both lipofectamine 2000 and 3000 and always get the same results. The lenti293t cell line has been tested as a suitable cell line for lentiviral production due to high transfection efficiency, high level of protein yield and high titer of lentiviral production. Lipofectamine 3000 has two main advantages over our existing reagent, the biggest one is its significantly higher transfection efficiency in difficult to transfect cell lines.
See page 2 to view a typical dna transfection procedure. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. 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. The intracellular trafficking mechanism of lipofectaminebased. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Ive seen it with lipofectamine 2000 or by transfection with polyethylenimine pei. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of. You can find in the instruction manuals of these two reagents a simple way of doing so. 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. Transfection protocol of lipofectamine rnaimax can be. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Lipofectamine 2000 transfection reagent from thermo fisher.
Lipofectamine 2000 is commonly used for sirna transfections. To perform transfection experiments in other cell culture plates. Mcf7, breast cancer, human, lipofectamine 2000 reagent. Can anyone please explain why we need the p3000 in addition to lipofectamine or how p3000 helps in adition to the lipo.
Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. A sample protocol is listed here for transfection experiments performed in 6well plates. I follow the standard recommended testing procedures and protocol for transfection. For additional information on scaling your transfection reaction, see page 4. Both lipofectamine 2000 and 3000 work well for 293t cells, you can just follow their protocol. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. Lipidbased transfection reagents exhibit cryoinduced increase in. Lipofectamine plasmid transfection bridges lab protocols. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Genscript recommends using lipofectamine 2000 for all transfections.
Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic. Superior performance for co transfection of sirna and plasmid dna. I want to try to use hela cell and lipofectamine 2000 to do. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. But muscle, for example, will be hard to transfect. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. The lipofectamine 3000 is the bees knees though, i managed to get loads.
Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. The reagent provides high transfection efficiency in many cells types and formats. Trachea smooth muscle, muscle, cow, lipofectamine reagent. The intracellular trafficking mechanism of lipofectamine.
Comparison of three different transfection methods biology. For more tips for your rnai experiment, refer to seven steps to rnai success. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection. This protocol is for transfecting plasmid dna into mammalian cells. From the luciferase activities and consistencies of data using different transfection reagents including genejammer, effectine, wellfect, lipofectamine, what i can say from the data i observed, the efficiency of transfection is much more better in hct116 cells using lipofectamine 2000. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market.
Viafect transfection reagent is a highperforming, lowtoxicity reagent. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. After 4h aspirate media and refeed cells with normal media. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Graphpad prism software and students ttest was used to. Troubleshooting for transfection of the crisprcas9 using. Viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
Lipofectamine is formulated to give high transfection. How does transfection with lipofectamine 2000 work. Cellspecific transfection protocols thermo fisher scientific us. Lipofectamine 2000 transfection protocol 10cm plates reagents. However, few studies have examined cellular responses to this delivery system. 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. Invitrogen lipofectamine 2000 transfection reagent life. 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. Do any one has protocol for stable transfection with lipofectamine 2000. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of.