Lipofectamine 2000 transfection protocol 10cm plates reagents. Enable high transfection efficiency in novel genome editing applications. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. 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. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Lipofectamine 2000 transfection reagent thermo fisher scientific. Make sure that cells are greater than 90% confluent and vary dna. The product manual includes advice on how to optimize transfection efficiency, as a number of. I would like to transfect 293t cells with lipofectamine 3000. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells.
Lipofectamine 3000 thermo fisher lipofectamine 3000 10. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Manual solid phase peptoid synthesis of liptoid was conducted according to. In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over 50,000 references to date. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Lipofectamine 2000 dna transfection reagent protocol.
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 trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8. Preliminary study of a novel transfection modality for in vivo sirna. Transfection was measured by luciferase activity using a conventional assay. Systematic screening of commonly used commercial transfection. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Fectopro transfection reagent for bioproduction polyplus. To transfect cells with sirna, follow the protocol as described for dna but. 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. Stealthsirna transfection protocol lipofectamine 2000. Lipofectamine plasmid transfection bridges lab protocols.
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. Genscript recommends using lipofectamine 2000 for all transfections. I found this possibility in lipofectamine 2000 manual, but not in lipofectamine 3000 manual. Lipofectaminez 2000 for transfection provides the following advantages. Lipofectamine transfection of c2c12 cells bridges lab. Cells in a 6 well dish, plated and at 70% confluence. Invitrogen lipofectamine 3000 transfection reagent. More importantly, on chip high throughput virus production combined with in situ target cell infection was achieved.
Lipofectamine, lipofectamine 2000, and lipofectamine enhan. Prewarm 50ml of optimum stored in cold room at 4c 10min. Anyone else have toxicity issues with lipofectamine 2000. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Cells in a 6 well dish, plated and at 90% confluence. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. How does transfection with lipofectamine 2000 work. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays. Lipofectamine 3000 showed the third highest transfection efficacy 37. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Moreover, there is a high risk of degradation in complete serum if nucleic acids are not well protected.
Maintain p19 cells undifferentiated in mem with 10% serum 7. Cells should be plated on collagencoated dishes, as for normal propagation. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. 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 transfected. Lipofectamine 2000 is a very efficient transfection agent, the stability of your knockdown or your overexpression is only related to the half life of your sirna, plamid in the cell. 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. Higher densities can lead to spontaneous differentiation. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. 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. Antibodies secondary antibodies elisa kits assay kits biomolecules. Lipofectamine 2000 transfection reagent dartmouth college. Highest transfection efficiency in many cell types and formats e. Freestyle 293f cells were transfected at a cell density of 1 x 106 cellsml in a 60 ml shaker flask using 293fectin and the freestyle protocol.
Optimization of transfection methods for huh7 and vero. Lipofectamine 3000 reagent was developed for high efficiency transfection and improved cell viability in the broadest range of cell types. While, the wet method has higher transfection efficiency as the premixing of dna with the transfection reagent lipofectamine 2000 facilitates the formation of the dnaliposome complex. High gfp expression following transfection using lipofectamine 2000 reagent. Invitrogen lipofectamine 3000 transfection reagent 0. Lipofectamine reagent recognition publications citing lipofectamine 2000 reagent steadily rose after the market introduction in 1999, and this product continues to be one of the mostcited transfection reagents on. The transfection efficiencies of the pte cells were 5% effectene. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine 2000 transfection reagent from invitrogen.
B hela cells were transfected with luciferase encoding plasmid dna using either transit. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine, a popular in vitro transfection reagent composed of cationic lipid. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Does lipofectamine 2000 give a stable transfection.
Generally need to have 2 g of dna for each tranfection. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Reverse transfection is the transfer of genetic material into cells and is reverse because the order of dna and cells is reverse that of conventional transfection. 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.
Effects of lipofectamine 2000sirna complexes on autophagy. Add 6l of lipofectamine2000 from biostores and 100 l of. I use 24well plates for transfections in triplicates. We appreciate greatly the technical service of thermofisher and find them an. All transit dna transfection reagents, including transitx2 dynamic delivery system, can be used for reverse transfection of dna into multiple cell types. For gene silencing, lipofectamine 2000 transfection reagents highefficiency transfections provide the high levels of gene knockdown needed to produce. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Effects of lipofectamine 2000sirna complexes on autophagy in hepatoma cells. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. For adherent cells, we recommend 9095% confluence at the time of transfection for high efficiency, high.
Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Invitrogen lipofectamine 3000 transfection reagent from. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. The mechanism of lipofectamine 2000 mediated transmembrane. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. For all transfection of multiple wells in 6 well plate, multiply cdna, optimem, and lipofectamine quantity used by number of wells to transfect. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best. 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. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Following transfection the cells were cultured in 25 ml serum containing medium. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates.
This protocol is for transfecting plasmid dna into mammalian cells. Other thing is to incease the cell density to 8595% confluncy as suggested by the manufacturer. In fig 1 below there is very high level of low level transfection and there are also high levels of high. Advanced transfection with lipofectamine 2000 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. Despite this, a satisfactory mechanismbased explanation of their. Department of neurobiology and anatomy, university of utah school of medicine, salt lake city, utah. Thee two most important factors in choosing transfecting reagent are.
Comparison of three different transfection methods biology. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Branski rc,0000000311909036, nyu school of medicine. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone.
Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. From the above data in table 3, this indicates that the 1. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. It consistently produces high transfection efficiency and high protein overexpression. Max % of positive cells after transfection of a gfpplasmid 3. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations.
Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Lipofectamine reagent recognition publications citing lipofectamine 2000 reagent steadily rose after the market introduction in 1999, and this product continues to be one of the mostcited transfection reagents on the market figure 3. For 96 well plate for me worked well plating approximately cells well transfecting them with 300ng dna and 0.
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