The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Lipofectamine 2000 transfection reagent dartmouth college. The day prior to your transfection, seed your cells so that they will be 70% to 90% confluent at the time of your experiment. 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. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. How does transfection with lipofectamine 2000 work. Versatileone reagent for dna, rna, and cotransfection.
Lipofectamine 2000 transfection reagent from invitrogen. Genscript recommends using lipofectamine 2000 for all transfections. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. I want to try to use hela cell and lipofectamine 2000 to do. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. The mechanism of lipofectamine 2000 mediated transmembrane. Viafect transfection reagent transfection assay e4981. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3.
Original article an improved method for increasing the efficiency of. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. I have used both lipofectamine 2000 and 3000 and always get the. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Anyone else have toxicity issues with lipofectamine 2000. Dna complexes were added to cells that had already been allowed to. 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 2000 is commonly used for sirna transfections. Transfection was measured by luciferase activity using a conventional assay.
Invitrogen lipofectamine 3000 transfection reagent from. The transfection efficiencies of the pte cells were 5% effectene. What percentage confluency should be used for stable transfection. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. It is used to increase the transfection efficiency of rna. Add 6l of lipofectamine2000 from biostores and 100 l of. Despite this, a satisfactory mechanismbased explanation of their. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates.
Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual. Lipofectamine plasmid transfection bridges lab protocols.
Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. 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.
Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Max % of positive cells after transfection of a gfpplasmid 3. Plate cells so they will be 7090% confluent at the time of transfection. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. 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. Protocol for human skeletal muscle myoblasts transfection. Cells in a 6 well dish, plated and at 70% confluence. Transfection protocol of lipofectamine rnaimax can be. Each tube gets 8mls of optimem, 32ug of gag pol, 12. Viafect transfection reagent is a highperforming, lowtoxicity reagent. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells.
Invitrogen lipofectamine 3000 transfection reagent. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. I am about to start my stable transfections with lipofectamine 2000. Highest transfection efficiency in many cell types and formats e. 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.
Systematic screening of commonly used commercial transfection. I want to try to use hela cell and lipofectamine 2000 to. 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. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transient transfection of mammalian cells with lipofectamine. To perform transfection experiments in other cell culture plates, simply multiply the. I use 24well plates for transfections in triplicates. Viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l. Lipofectamine is formulated to give high transfection efficiency with little. The media needs to be changed at least 1hr prior to transfection. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. In tube a place 2 g of dna in optimemi for a final volume of 200 l. I have used both lipofectamine 2000 and 3000 and always get the same results.
After 4h aspirate media and refeed cells with normal media. For additional information on scaling your transfection reaction, see page 4. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Do any one has protocol for stable transfection with lipofectamine 2000. The intracellular trafficking mechanism of lipofectamine. Refer to the cell lines database for a list of cell types successfully transfected. A sample protocol is listed here for transfection experiments performed in 6well plates. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 dna transfection reagent protocol. Prewarm 50ml of optimum stored in cold room at 4c 10min. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Cellspecific transfection protocols thermo fisher scientific us. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. This protocol is for transfecting plasmid dna into mammalian cells. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery. Invitrogen lipofectamine 2000 transfection reagent. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. However, few studies have examined cellular responses to this delivery system. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Highest transfection efficiency in many cell types and formats. The nucleofection resulted in a transfection efficiency of 90% and cytotoxicity of 5%.
The lenti293t cell line has been tested as a suitable cell line for lentiviral production. 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. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Refer to the cell lines database for a list of cell types successfully. Protocol for human skeletal muscle myoblasts hsmm transfection by fugene hd. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. 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. Effects of lipofectamine 2000sirna complexes on autophagy. Comparison of three different transfection methods biology.
Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Lipofectamine transfection of c2c12 cells bridges lab. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. 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. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
Advanced transfection with lipofectamine 2000 reagent. Lipofectamine 2000 transfection protocol 10cm plates reagents. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Lipofectamine 3000 reagent thermo fisher scientific tr. Troubleshooting for transfection of the crisprcas9 using. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent.
Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Combine the two volumes of optimemdna and optimemlipofectamine. Mscs, spinal muscular atrophy patient fibroblasts sma, human hepatoma hepg2. The intracellular trafficking mechanism of lipofectaminebased. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Invitrogen lipofectamine transfection reagent protocols have been. An improved method for increasing the efficiency of gene. May 11, 2016 for te experiments, lipofectaminedna and dotapdopcdna complexes were prepared in optimem invitrogen by mixing for each well of 24well plates 0.
Effects of lipofectamine 2000sirna complexes on autophagy in. 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. Comparative transfection of dna into primary and transformed. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature.
The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Other thing is to incease the cell density to 8595% confluncy as suggested by the manufacturer. General procedure for transfection of mammalian cells with. 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. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. 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.