For additional information, and protocols on transfecting suspension. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 3000 protocol written out with timing. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. In addition, invitrogen claims that lipofectamine 2000 gives the most proven results in more functional and phenotypic assays than any other reagent and they back up this claim with an abundance of references. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene exp. Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates 1 mix 0. Protocol 6 well dish plate cells the day before so that they are at 9095% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg.
Protocol for lipofectamine transfection in nih3t3 cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Invitrogen lipofectamine 2000 transfection reagent life. Lipofectamine 2000 transfection reagent from thermo fisher. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Invitrogen lipofectamine 2000 transfection reagent fisher. A sample protocol is listed here for transfection experiments performed in 6well plates. Advanced transfection with lipofectamine 2000 reagent. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna.
Lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine plasmid transfection bridges lab protocols. Service, as are celltype specific rnai transfection protocols see rnai protocols. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
Superior performance for cotransfection of sirna and plasmid dna with reliable performance for highthroughput applications. Transfection efficiencies lower than 50% were not reported in this table. Materials puromycin dihydrochloride life technologies, cat. 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. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Lipofectamine 3000 reagent thermo fisher scientific. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine transfection reagent is one of our first generation cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells.
Highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 transfection reagent is a proprietary formulation. 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 is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Invitrogen lipofectamine 2000 transfection reagent 0. Invitrogen lipofectamine 2000 transfection reagent 1. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Refer to the cell lines database for a list of cell types successfully.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Despite this, a satisfactory mechanismbased explanation of their. Ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems. Lipofectamine 2000 transfection reagent dartmouth college. I performed lipofectamine2000 transfection on hela cells and got really good results checked transient transfection under fluorescence microscope and had very high efficiency. Dharmafect 1 transfection reagent horizon discovery.
Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Lipofectamine 2000 reagent thermo fisher scientific. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number.
Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Transfection protocol transfection components component cat. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Invitrogen lipofectamine 3000 transfection reagent 0. I am performing transfection on mcf7 cells using lipofectamine 3000 invitrogen but unable to get any transfectants, instead the cells are dying after incubation with reagent and dna. Invitrogen lipofectamine 3000 transfection reagent from. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish.
The intracellular trafficking mechanism of lipofectamine. Transfection protocol lipofectamine 3000 transfection reagent. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Transfection guide overview of transfection methods. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Exceptional transfection efficiency in a broad range of cell lines with high levels of recombinant protein expression and a simple protocol. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Invitrogens lipofectamine 2000 is highly cited in the literature, a sign of a successful reagent, so when our lab embarked upon the path to develop a successful transfection protocol, we included lipofectamine 2000 along with the other leading reagents.
Invitrogen lipofectamine rnaimax transfection reagent. So any one who has done can tell me the efficiency of transfection using lipofectamine. Lipofectamine 2000 is cationic based lipid delivery system, which transports dna into cells by fusing with plasma membrane. This protocol is well adjusted for cancer cell lines. To transfect cells with sirna, follow the protocol as. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine 3000 reagent for pluripotent stem cell applications. Lipofectamine 3000 transfection reagent hepg2 liver. Plate cells so they will be 6080% confluent at the time of transfection. Used the same reagent and methods including wash steps and plasmid on u87 cells and had very bad results high toxicity with normally adherent cells in suspension. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. See page 2 to view a typical plasmid transfection using a lipofectamine reagent. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types.
Life technologies corporation andor its affiliates warrant their products as set forth in the life. 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. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. You continue to choose these reagents due to their wide acceptance as the best and most reliable available. Lipofectamine 2000 from invitrogen biocompare product. You can generally use other serumfree medium instead of optimem. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. 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. Equal to or greater transfection efficiency compared with lipofectamine 2000 in sirna transfection figure 4. The trusted, simple, broadspectrum transfection reagent for most cell lines. 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 suggested quantities by the relative surface area of your plate. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Invitrogen s lipofectamine 2000 is highly cited in the literature, a sign of a successful reagent, so when our lab embarked upon the path to develop a successful transfection protocol, we included lipofectamine 2000 along with the other leading reagents. Since launching in 1993, invitrogen lipofectamine reagents have become the most referenced transfection reagents with over 50,000 citations to date. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. High transfection effi ciency enables lipofectamine 3000 to be used for a variety of applications, including reprogramming of fi broblasts, gene editing, and transfection of diffi cult cells. 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. Endofectin transfection reagent, cell lines genecopoeia. Transfection protocol lipofectamine 3000 transfection. Invitrogen lipofectamine 3000 transfection reagent fisher.
Lipofectamine 3000 transfection reagent kit youtube. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Invitrogen lipofectamine 2000 transfection reagent is a proprietary. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 transfection reagent from invitrogen. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. Lipofectamine 2000 reagent protocol thermo fisher scientific. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that. Superior transfection efficiency for a broad range of cell lines compared with lipofectamine 2000 figures 2 and 3.
Latter i have planned to do transfection experimetns using lipofectamine in 24 well plate. Plate cells so they will be 7090% confluent at the time of transfection. Refer to the cell lines database for a list of cell types successfully transfected. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Can anyone provide me with a proper transfection protocol. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects.