Invitrogen lipofectamine 2000 transfection reagent. Despite this, a satisfactory mechanismbased explanation of their. Highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 dna transfection reagent protocol.
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. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine transfection reagent is one of our first generation cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. Lipofectamine 3000 protocol written out with timing specifics. This protocol is well adjusted for cancer cell lines. Invitrogen lipofectamine 3000 transfection reagent 0. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems. Refer to the cell lines database for a list of cell types successfully. Prewarm 50ml of optimum stored in cold room at 4c 10min.
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. For additional information, and protocols on transfecting suspension. You can generally use other serumfree medium instead of optimem. 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. To transfect cells with sirna, follow the protocol as. Lipofectamine 3000 transfection reagent kit youtube. Add 6l of lipofectamine2000 from biostores and 100 l of. 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 reagent thermo fisher scientific. Lipofectamine 2000 transfection reagent from thermo fisher. Dharmafect 1 transfection reagent horizon discovery.
Refer to the cell lines database for a list of cell types successfully transfected. Lipofectamine 2000 transfection reagent is a proprietary formulation. Materials puromycin dihydrochloride life technologies, cat. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0.
Plate cells so they will be 6080% confluent at the time of transfection. Transfection efficiencies lower than 50% were not reported in this table. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Lipofectamine 2000 transfection reagent from invitrogen. Invitrogen lipofectamine 2000 transfection reagent fisher. Lipofectamine 2000 reagent protocol thermo fisher scientific.
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. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. So any one who has done can tell me the efficiency of transfection using lipofectamine. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Protocol for lipofectamine transfection in nih3t3 cells using cells plated in 8well plates 1 mix 0.
Endofectin transfection reagent, cell lines genecopoeia. Lipofectamine 3000 protocol written out with timing. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of.
Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. Lipofectamine plasmid transfection bridges lab protocols. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. 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. 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 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays.
Invitrogen lipofectamine 2000 transfection reagent is a proprietary. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfection protocol lipofectamine 3000 transfection reagent. You continue to choose these reagents due to their wide acceptance as the best and most reliable available. Transfection guide overview of transfection methods. Lipofectamine 3000 reagent thermo fisher scientific. 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. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. 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. 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.
Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Service, as are celltype specific rnai transfection protocols see rnai protocols. Outline of protocol steps for the use of lipofectamine 2000 reagent. Invitrogen lipofectamine 3000 transfection reagent. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. Lipofectamine transfection reagent from thermo fisher. 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 versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. Invitrogen lipofectamine 2000 transfection reagent 1.
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. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine 2000 invitrogen how much can transient transfection expected. 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. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna. 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. Superior performance for cotransfection of sirna and plasmid dna with reliable performance for highthroughput applications. Plate cells so they will be 7090% confluent at the time of transfection. 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.
Transfection protocol lipofectamine 3000 transfection. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 transfection protocol 10cm plates reagents. The trusted, simple, broadspectrum transfection reagent for most cell lines. Life technologies corporation andor its affiliates warrant their products as set forth in the life. Exceptional transfection efficiency in a broad range of cell lines with high levels of recombinant protein expression and a simple protocol. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene exp.
For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Invitrogen lipofectamine 2000 transfection reagent 0. Can anyone provide me with a proper transfection protocol. Lipofectamine 2000 is cationic based lipid delivery system, which transports dna into cells by fusing with plasma membrane. Lipofectamine 2000 from invitrogen biocompare product. Invitrogen lipofectamine 3000 transfection reagent fisher. Invitrogen lipofectamine 3000 transfection reagent from. Lipofectamine 2000 transfection reagent dartmouth college. The intracellular trafficking mechanism of lipofectamine. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. A sample protocol is listed here for transfection experiments performed in 6well plates. Protocol for lipofectamine transfection in nih3t3 cells.
Lipofectamine 2000 transfection reagent life technologies, cat. Invitrogen lipofectamine 2000 transfection reagent life. I performed lipofectamine2000 transfection on hela cells and got really good results checked transient transfection under fluorescence microscope and had very high efficiency. 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. Genscript recommends using lipofectamine 2000 for all transfections. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.
Superior transfection efficiency for a broad range of cell lines compared with lipofectamine 2000 figures 2 and 3. Lipofectamine 3000 reagent for pluripotent stem cell applications. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. See page 2 to view a typical plasmid transfection using a lipofectamine reagent. Lipofectamine 3000 transfection reagent hepg2 liver. Latter i have planned to do transfection experimetns using lipofectamine in 24 well plate.
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