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. 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. Invitrogen lipofectamine 3000 transfection reagent fisher. Lipofectamine 3000 transfection reagent kit youtube. Lipofectamine transfection reagent from thermo fisher. Lipofectamine 2000 transfection protocol 10cm plates reagents. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Invitrogen lipofectamine 3000 transfection reagent from. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Advanced transfection with lipofectamine 2000 reagent. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Prewarm 50ml of optimum stored in cold room at 4c 10min.
Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna. Invitrogen lipofectamine 2000 transfection reagent is a proprietary. Superior transfection efficiency for a broad range of cell lines compared with lipofectamine 2000 figures 2 and 3. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene exp. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. 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.
For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates 1 mix 0. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. Service, as are celltype specific rnai transfection protocols see rnai protocols. Lipofectamine 2000 from invitrogen biocompare product. 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. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Transfection protocol lipofectamine 3000 transfection reagent.
Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Genscript recommends using lipofectamine 2000 for all transfections. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. Highest transfection efficiency in many cell types and formats e. Exceptional transfection efficiency in a broad range of cell lines with high levels of recombinant protein expression and a simple protocol. The intracellular trafficking mechanism of lipofectamine. Lipofectamine plasmid transfection bridges lab protocols. Materials puromycin dihydrochloride life technologies, cat. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps.
Equal to or greater transfection efficiency compared with lipofectamine 2000 in sirna transfection figure 4. Transfection protocol lipofectamine 3000 transfection. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine 2000 transfection reagent life technologies, cat. You can generally use other serumfree medium instead of optimem. Invitrogen lipofectamine 2000 transfection reagent 1. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000 transfection reagent is a proprietary formulation. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. I performed lipofectamine2000 transfection on hela cells and got really good results checked transient transfection under fluorescence microscope and had very high efficiency. A sample protocol is listed here for transfection experiments performed in 6well plates.
Since launching in 1993, invitrogen lipofectamine reagents have become the most referenced transfection reagents with over 50,000 citations to date. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Outline of protocol steps for the use of lipofectamine 2000 reagent. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
The trusted, simple, broadspectrum transfection reagent for most cell lines. Endofectin transfection reagent, cell lines genecopoeia. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 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 2000 transfection reagent. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Latter i have planned to do transfection experimetns using lipofectamine in 24 well plate. Invitrogen lipofectamine 2000 transfection reagent 0. For additional information, and protocols on transfecting suspension. So any one who has done can tell me the efficiency of transfection using lipofectamine.
Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. See page 2 to view a typical plasmid transfection using a lipofectamine reagent. Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Transfection of nih3t3 cells, hela, swis 3t3, 293t 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. Refer to the cell lines database for a list of cell types successfully. Lipofectamine 3000 reagent thermo fisher scientific. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Lipofectamine 3000 protocol written out with timing.
Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of. Plate cells so they will be 7090% confluent at the time of transfection. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. Transfection efficiencies lower than 50% were not reported in this table.
Lipofectamine 2000 dna transfection reagent protocol. 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. Can anyone provide me with a proper transfection protocol. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Add 6l of lipofectamine2000 from biostores and 100 l of. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. 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. 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. Protocol for lipofectamine transfection in nih3t3 cells. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Invitrogen lipofectamine rnaimax transfection reagent. Despite this, a satisfactory mechanismbased explanation of their.
Lipofectamine 3000 protocol written out with timing specifics. 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. To transfect cells with sirna, follow the protocol as. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. 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. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Invitrogen lipofectamine 2000 transfection reagent fisher. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Invitrogen lipofectamine 2000 transfection reagent life. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. 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. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems.
Invitrogen lipofectamine 3000 transfection reagent. Lipofectamine 2000 reagent thermo fisher scientific. 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. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 transfection reagent from invitrogen. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Dharmafect 1 transfection reagent horizon discovery. Refer to the cell lines database for a list of cell types successfully transfected. This protocol is well adjusted for cancer cell lines. Lipofectamine 2000 is cationic based lipid delivery system, which transports dna into cells by fusing with plasma membrane. Lipofectamine 2000 invitrogen how much can transient transfection expected.
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. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Lipofectamine 2000 transfection reagent from thermo fisher. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Transfection guide overview of transfection methods. Invitrogen lipofectamine 3000 transfection reagent 0. Plate cells so they will be 6080% confluent at the time of transfection. You continue to choose these reagents due to their wide acceptance as the best and most reliable available. Transfection protocol transfection components component cat.
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