Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Advanced transfection with lipofectamine 2000 reagent. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. I am about to start my stable transfections with lipofectamine 2000. Lipofectamine transfection of c2c12 cells bridges lab. Protocol for human skeletal muscle myoblasts hsmm transfection by fugene hd. 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. Other thing is to incease the cell density to 8595% confluncy as suggested by the manufacturer. Lipofectamine 3000 reagent thermo fisher scientific tr. The mechanism of lipofectamine 2000 mediated transmembrane.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Plate cells for 212hrs but not over 24 hours prior to transfection to. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. May 11, 2016 for te experiments, lipofectaminedna and dotapdopcdna complexes were prepared in optimem invitrogen by mixing for each well of 24well plates 0. Protocol for human skeletal muscle myoblasts transfection. The day prior to your transfection, seed your cells so that they will be 70% to 90% confluent at the time of your experiment. Cellspecific transfection protocols thermo fisher scientific us.
Anyone else have toxicity issues with lipofectamine 2000. The nucleofection resulted in a transfection efficiency of 90% and cytotoxicity of 5%. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. I want to try to use hela cell and lipofectamine 2000 to do. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. It is used to increase the transfection efficiency of rna. Lipidbased transfection reagents exhibit cryoinduced increase in. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. To perform transfection experiments in other cell culture plates, simply multiply the.
What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual. Highest transfection efficiency in many cell types and formats. Comparison of three different transfection methods biology. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Aug 07, 2012 the day prior to your transfection, seed your cells so that they will be 70% to 90% confluent at the time of your experiment. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. How does transfection with lipofectamine 2000 work. In the liposome transfection, the quantity of lipofectamine 2000 could be reduced even further and this may produce a higher level of expression as lipofectamine can be toxic to cells. 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, intracellular. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Plate cells so they will be 7090% confluent at the time of transfection. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. 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. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Refer to the cell lines database for a list of cell types successfully transfected. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery.
Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Do any one has protocol for stable transfection with lipofectamine 2000. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. I have used both lipofectamine 2000 and 3000 and always get the. Dna complexes were added to cells that had already been allowed to. Invitrogen lipofectamine 2000 transfection reagent. Versatileone reagent for dna, rna, and cotransfection.
Transient transfection of mammalian cells with lipofectamine. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. Lipofectamine 2000 reagent thermo fisher scientific. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. What percentage confluency should be used for stable transfection.
Transfection was measured by luciferase activity using a conventional assay. 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. Effects of lipofectamine 2000sirna complexes on autophagy in. Prewarm 50ml of optimum stored in cold room at 4c 10min. Transfection protocol of lipofectamine rnaimax can be. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Add 6l of lipofectamine2000 from biostores and 100 l of. Comparative transfection efficiency of jetprime versus its main competitor. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. 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 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Lipofectamine plasmid transfection bridges lab protocols. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Despite this, a satisfactory mechanismbased explanation of their. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. However, few studies have examined cellular responses to this delivery system. 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. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1.
Cells in a 6 well dish, plated and at 70% confluence. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. An improved method for increasing the efficiency of gene. Troubleshooting for transfection of the crisprcas9 using. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. Lipofectamine 2000 is commonly used for sirna transfections. Effects of lipofectamine 2000sirna complexes on autophagy.
Highest transfection efficiency in many cell types and formats e. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Invitrogen lipofectamine transfection reagent protocols have been. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. Genscript recommends using lipofectamine 2000 for all transfections. 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.
Lipofectamine is formulated to give high transfection efficiency with little. The media needs to be changed at least 1hr prior to transfection. A sample protocol is listed here for transfection experiments performed in 6well plates. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection. Lipofectamine 2000 transfection reagent dartmouth college. Viafect transfection reagent is a highperforming, lowtoxicity reagent. Max % of positive cells after transfection of a gfpplasmid 3. General procedure for transfection of mammalian cells with.
Lipofectamine 2000 dna transfection reagent protocol. Each tube gets 8mls of optimem, 32ug of gag pol, 12. I use 24well plates for transfections in triplicates. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure.
The intracellular trafficking mechanism of lipofectamine. The transfection efficiencies of the pte cells were 5% effectene. Cells were transfected with a firefly luciferase encoding plasmid dna 0. I want to try to use hela cell and lipofectamine 2000 to. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. For additional information on scaling your transfection reaction, see page 4. This protocol is for transfecting plasmid dna into mammalian cells.
The lenti293t cell line has been tested as a suitable cell line for lentiviral production. Lipofectamine transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Mscs, spinal muscular atrophy patient fibroblasts sma, human hepatoma hepg2. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Original article an improved method for increasing the efficiency of. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. After 4h aspirate media and refeed cells with normal media.
Viafect transfection reagent transfection assay e4981. Refer to the cell lines database for a list of cell types successfully. The intracellular trafficking mechanism of lipofectaminebased. Lipofectamine 2000 transfection reagent from invitrogen. Invitrogen lipofectamine 3000 transfection reagent from. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Comparative transfection of dna into primary and transformed. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Systematic screening of commonly used commercial transfection. Invitrogen lipofectamine 3000 transfection reagent. I have used both lipofectamine 2000 and 3000 and always get the same results.
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