I have used both lipofectamine 2000 and 3000 and always get the same results. The impact of transfection mediated toxicity mirus bio. A sample protocol is listed here for transfection experiments performed in 6well plates. Do any one has protocol for stable transfection with lipofectamine 2000. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. 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. Invitrogen lipofectamine 2000 transfection reagent fisher. Invitrogen lipofectamine 2000 transfection reagent 15ml.
The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Can anyone please explain why we need the p3000 in addition to lipofectamine or how p3000 helps in adition to the lipo. Cellspecific transfection protocols thermo fisher scientific us. 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. 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.
Transient transfection of mammalian cells with lipofectamine. Invitrogen lipofectamine transfection reagent protocols have been. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. Comparison of three different transfection methods biology. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. The intracellular trafficking mechanism of lipofectaminebased. Invitrogen lipofectamine 2000 transfection reagent life. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. From the above data in table 3, this indicates that the 1. Ive seen it with lipofectamine 2000 or by transfection with polyethylenimine pei.
Research diagnostic development diagnostic testing precision medicine preclinical to companion diagnostic development public health. Highest transfection efficiency in many cell types and formats e. Lipofectamine is formulated to give high transfection. Viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l. Lipofectamine 2000 transfection reagent from thermo fisher. I am getting shitty transfection efficiency using effectene and geneporter 2 transfection reagent. Lipofectamine 3000 transfection reagent kit youtube. Systematic screening of commonly used commercial transfection.
Invitrogen lipofectamine 2000 transfection reagent. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. To perform transfection experiments in other cell culture plates. The company has no samples for test, so, i would like to know if anyone can give a feedback on how they compare. Trachea smooth muscle, muscle, cow, lipofectamine reagent. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number.
Add 6l of lipofectamine2000 from biostores and 100 l of. Does anyone have experience with the lipofectamine 2000. After 4h aspirate media and refeed cells with normal media. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of. The lenti293t cell line has been tested as a suitable cell line for. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. I follow the standard recommended testing procedures and protocol for transfection. The manual recommends days whereas some people transfect for a 4. I want to try to use hela cell and lipofectamine 2000 to do transfection. But muscle, for example, will be hard to transfect.
However, few studies have examined cellular responses to this delivery system. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. 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. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. 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. Lipofectamine plasmid transfection bridges lab protocols. What percentage confluency should be used for stable transfection. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity.
How does transfection with lipofectamine 2000 work. Effects of lipofectamine 2000sirna complexes on autophagy in. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. 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. 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. Transfection protocol of lipofectamine rnaimax can be. 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. Combine the two volumes of optimemdna and optimemlipofectamine. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection.
The lipofectamine 3000 is the bees knees though, i managed to get loads. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. Plate cells for 212hrs but not over 24 hours prior to. The mechanism of lipofectamine 2000 mediated transmembrane.
For additional information on scaling your transfection reaction, see page 4. The intracellular trafficking mechanism of lipofectamine. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Studies were undertaken to evaluate and compare the transfection efficacy. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3.
Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. See page 2 to view a typical dna transfection procedure. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Both lipofectamine 2000 and 3000 work well for 293t cells, you can just follow their protocol. Help on transfecting mcf7 and hct 116 cells transfection.
I want to try to use hela cell and lipofectamine 2000 to. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following. This protocol is for transfecting plasmid dna into mammalian cells. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Troubleshooting for transfection of the crisprcas9 using lipofectamine. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. Lipofectamine 2000 is commonly used for sirna transfections. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Effects of lipofectamine 2000sirna complexes on autophagy.
You can find in the instruction manuals of these two reagents a simple way of doing so. Examples of cells transfected successfully thermo fisher scientific. Lipofectamine transfection of c2c12 cells bridges lab. Invitrogen lipofectamine 3000 transfection reagent from.
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. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically. 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. An improved method for increasing the efficiency of gene.
Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. As always, use good cell culture practices and wear your personal protective. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine 2000 reagent thermo fisher scientific. Does anyone have experience with the lipofectamine 2000 toxicity. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. 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. 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. Clinical laboratory products microsatellite instability testing. 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. Original article an improved method for increasing the efficiency of. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays.
Troubleshooting for transfection of the crisprcas9 using. Superior performance for co transfection of sirna and plasmid dna. The trusted, simple, broadspectrum transfection reagent for most cell lines. For more tips for your rnai experiment, refer to seven steps to rnai success. Start any new transfection by testing the recommended four. Lipofectamine 2000 dna transfection reagent protocol.
Lipidbased transfection reagents exhibit cryoinduced increase in. Viafect transfection reagent is a highperforming, lowtoxicity reagent. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell. I am about to start my stable transfections with lipofectamine 2000. Enhanced transfection of cryomanipulated lf2000 is not dependent on a specific promoter. 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. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Lipofectamine 2000 transfection protocol 10cm plates reagents. Mcf7, breast cancer, human, lipofectamine 2000 reagent.
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. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. I use 24well plates for transfections in triplicates. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of. General procedure for transfection of mammalian cells with. Different cell confluencies, dnareagent ratios and total transfection volumes were optimized for two chemical reagents including jetpei and lipofectamine 2000. I want to try to use hela cell and lipofectamine 2000 to do.
Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Graphpad prism software and students ttest was used to. Viafect transfection reagent transfection assay e4981. Statistical significance was evaluated by a twotailed students ttest. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection.
Advanced transfection with lipofectamine 2000 reagent. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Prewarm 50ml of optimum stored in cold room at 4c 10min. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338 500 pdf. 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. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic. 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. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.
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