Add 6l of lipofectamine2000 from biostores and 100 l of. Mouse neuronal cells were transfected with a gfpexpressing plasmid using either primefect transfection reagent c, or lipofectamine 2000 d. 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. Highest transfection efficiency in many cell types and formats e. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Our reagents are ideal for delivering multiple types of nucleic acids including. The reagent provides high transfection efficiency in many cells types and formats. Comparison of primefect tm and lipofectamine 2000 in plasmid transfection on mouse neuronal cells. I want to try to use hela cell and lipofectamine 2000 to. After determining the optimum reagent amount, use the multiplication factor to determine the reagent amount needed for your new plate format. For additional information, and protocols on transfecting suspension cells refer to the manual at. Invitrogen lipofectamine 3000 transfection reagent fisher. For additional information on scaling your transfection reaction, see page 4.
Cells were seeded in 96well flat bottom nontissue culture plate just before transfection with the density 50,000 cells per well in 100. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. Use a range of 20100 nm stealth rnai or sirna for 24well format. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Lipofectamine 2000 transfection reagent from thermo fisher. To transfect cells in different tissue culture formats, vary. Refer to the cell lines database for a list of cell types successfully transfected. Dec 31, 20 lipofectamine 3000 has two main advantages over our existing reagent, the biggest one is its significantly higher transfection efficiency in difficult to transfect cell lines.
Transfection of mammalian cells using linear polyethylenimine. I want to try to use hela cell and lipofectamine 2000 to do. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transfection protocol lipofectamine 3000 transfection reagent ncih1299 lung cancer cells complete growth medium component cat. Lipofectamine 3000 transfection reagent kit youtube. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Invitrogen lipofectamine 3000 transfection reagent l3000008. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Transfection efficiencies lower than 50% were not reported in this table. Lipofectamine 2000 transfection reagent dartmouth college. Dilute dna in 250 l of optimem i reduced serum medium without serum or other medium without serum.
Transfection protocol of lipofectamine rnaimax can be. Each reagent was tested with a range of transfection reagent. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Optimized conditions for successful transfection of human. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. The manual recommends also that a certain amount of nucleic acid, described in ug, should be used for transfection. Cells for transfection were harvested at approximately. In contrast, transfection of cells with lipofectamine 2000 mrna complexes in optimem i reduced serum media or the use of transit mrna transfection reagent mrna complexes in cell culture. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. I previously believed that the best method was using lipofectamine 2000 with plus reagent and an overnight incubation with optimem. Cells for transfection were grown in 15 ml cdcho medium gibco in 50 ml tubes on rotator at 110 rpm. Transfection protocol transfection components component cat.
Transfection transit transfection reagents mirus bio. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection. 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. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Lipofectamine 2000 dna transfection reagent protocol. Transfection guide overview of transfection methods promega. It is used to increase the transfection efficiency of rna. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with. Using lipofectamine 2000 cd reagent for transfection provides the following advantages.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Invitrogen lipofectamine 2000 transfection reagent 0. Trans it transfection reagents are designed, tested and manufactured in madison, wi. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. 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. 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.
Invitrogen lipofectamine 3000 transfection reagent. While lipofectamine 2000 reagent demonstrated transfection efficiencies only. Cloned genes can be transfected into cells for biochemical characterization. Transit lt1 transfection reagent is a broad spectrum reagent that provides high efficiency plasmid dna delivery in many mammalian cell types including primary cells. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from.
I previously believed that the best method was using. Depending on the nature of the target gene, you may. Transfection efficiency of p19 cells is between 3050%. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to transfect 17 cell lines with. It is best to use polypropylene instead of polystyrene tubes.
Transfection protocol lipofectamine 3000 transfection reagent gct. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo. We support multiple transfection methods with a different reagent for nearly any. Transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best performing polymeric nanoparticles. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies.
Lipofectamine 2000 reagent dna seed cells so they are 7090% con. Lipofectamine 2000 reagent thermo fisher scientific. Second, lipofectamine is inhibited by serum in the media and. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Table 2 examines transfection efficiency and cell viability under optimal conditions for each reagent 24 hours after transfection. For your convenience, the essential components of this protocol are now.
Mar 03, 2014 the transfection of ecs with stemfect rna transfection reagent and lipofectamine 2000 in cell culture medium resulted in very low levels of transfection. Depending on the nature of the target gene, you may transfect cells at higher densities. A sample protocol is listed here for transfection experiments performed in 6well plates. Genscript recommends using lipofectamine 2000 for all transfections. Comparison of transfection efficiency of nonviral gene transfer reagents.
May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine 2000 reagent protocol thermo fisher scientific. Fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. The transfection of ecs with stemfect rna transfection reagent and lipofectamine 2000 in cell culture medium resulted in very low levels of transfection. Outline of protocol steps for the use of lipofectamine 2000 reagent. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. We support multiple transfection methods with a different reagent for nearly any application.
Thee two most important factors in choosing transfecting reagent are. The microscopic analysis was performed using antirigi mab. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Lipofectamine 2000 transfection reagent from invitrogen. Cells for transfection were harvested at approximately 1,000,000 cells ml. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Choosing the right transfection reagent for optimal efficiency. Dna, sirna, mirna, mrna, viral rna, crisprcas9 components, and oligonucleotides to. On the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using lipofectamine 3000 transfection reagent. Comparative transfection efficiency of jetprime versus its main competitor. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance.
Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. The reagent provides high transfection efficiency in many cells types and. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Increase transfection efficiency and cell viability with. Comparison of transfection efficiency of nonviral gene. For each transfection sample, prepare dnalipofectamine 2000 complexes as follows. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Introduction 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. Invitrogen lipofectamine 2000 transfection reagent 1. Transfection protocol lipofectamine 3000 transfection. I have been doing research with primary rat cardiomyocytes for more than seven years, and plasmid transfection of these cells is not easy. Gibco rpmi 1640 medium with glutamax supplement 61870036 10% gibco fbs a3160401 25 mm gibco hepes 1 m 15630080 1. L cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0.
Second, lipofectamine is inhibited by serum in the media and, therefore, requires a change of the medium before and after the transfection. Transfection guide overview of transfection methods. A hela cells were transfected with hcv dsrna using lipofectamine 2000 reagent. Invitrogen lipofectamine 2000 transfection reagent life. It consistently produces high transfection efficiency and high protein overexpression. The product manual includes advice on how to optimize transfection efficiency, as a number of conditions can be varied including dna. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
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