Start any new transfection by testing the recommended four concentrations of lipofectamine2000 reagent to determine an optimum amount. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Transfection was performed using 4 l lipofectamine 2000 and 1. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. In tube a place 2 g of dna in optimemi for a final volume of 200 l. Lipofectamine 2000 transfection reagent is a superior choice for co transfection, given its effectiveness for transfecting both sirna and plasmid dna. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may. 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.
Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. I am about to start my stable transfections with lipofectamine 2000. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. For each transfection sample, prepare dnalipofectamine 2000 complexes as follows. Protocol and application notes back to table of content. Refer to the cell lines database for a list of cell types successfully. The mechanism of lipofectamine 2000 mediated transmembrane. Versatileone reagent for dna, rna, and cotransfection. Invitrogen lipofectamine 2000 transfection reagent life. Combine the two volumes of optimemdna and optimemlipofectamine.
Transfection was measured by luciferase activity using a conventional assay. The development of multiresistant strains of staphylococcus aureus is now a major public health hazard. See page 2 to view a typical dna transfection procedure. This protocol is for transfecting plasmid dna into mammalian cells. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Max % of positive cells after transfection of a gfpplasmid 3. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Maintain p19 cells undifferentiated in mem with 10% serum 7. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency.
An improved method for increasing the efficiency of gene. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. 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. Out of the five transfection systems tested, fugene and rnaimax. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1.
Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of. Add dnalipofectamine complexes 500 ul to the 2 ml of media on the cells. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Comparison of three different transfection methods biology.
Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Highest transfection efficiency in many cell types and formats. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. General procedure for transfection of mammalian cells with. A sample protocol is listed here for transfection experiments performed in 6well plates. The recent emergence of vancomycinresistant strains in multiple countries, including the united states, has further underscored the importance of identifying microbial factors that may lead to the development of preventive vaccines and novel antimicrobial therapy. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format. 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. For 96 well plate for me worked well plating approximately cells well transfecting them with 300ng dna and 0. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Advanced transfection with lipofectamine 2000 reagent. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent.
Cells in a 6 well dish, plated and at 70% confluence. Lipidbased transfection reagents exhibit cryoinduced increase in. Invitrogen lipofectamine 2000 transfection reagent. Dilute dna in 250 l of optimem i reduced serum medium without serum or other medium without serum. Among them, although rnaimax displayed the second highest transfection efficacy, and it had the best safety profile, with 83. The nucleofection resulted in a transfection efficiency of 90% and cytotoxicity of 5%. The mtt test for transfections with ptracer and prep9 without. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 2000 dna transfection reagent protocol. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Add 6l of lipofectamine2000 from biostores and 100 l of. 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. Lipofectamine 3000 reagent thermo fisher scientific fr.
Lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Mix lipofectamine 2000 gently before use, then dilute the appropriate amount in 250. 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. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Optimization of transfection methods for huh7 and vero. I have used both lipofectamine 2000 and 3000 and always get the same results. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over 50,000 references to date. Thee two most important factors in choosing transfecting reagent are. Invitrogen lipofectamine 3000 transfection reagent from.
For additional information on scaling your transfection reaction, see page 4. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Lipofectamine 2000 reagent thermo fisher scientific.
Cells were transfected with a firefly luciferase encoding plasmid dna 0. Lipofectamine transfection of c2c12 cells bridges lab. Invitrogen in serumfree dmem for 24 h according to manufacturer instructions. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. A gfp green fluorescent protein plasmid can be used to determine transfection efficiency. Troubleshooting for transfection of the crisprcas9 using. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. In the liposome transfection, the quantity of lipofectamine 2000 could be reduced even further and this may produce a higher level of expression as lipofectamine can be toxic to cells. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000 transfection reagent from invitrogen.
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. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. 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. Enhanced transfection of cryomanipulated lf2000 is not dependent on a specific promoter. Nucleofection is a highly effective gene transfer technique for human. Lipofectamine 3000, rnaimax, fugene and lipofectamine 2000 all displayed high transfection efficacy 97. The intracellular trafficking mechanism of lipofectamine. 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. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells.
The transfection efficiencies of the pte cells were 5% effectene. 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. Comparative transfection of dna into primary and transformed. What percentage confluency should be used for stable transfection. Lipofectamine plasmid transfection bridges lab protocols. Lipofectamine 2000 transfection protocol 10cm plates reagents. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Luciferase activity was measured after 24 hours figures 24. Transfection was performed as described in protocol 2 with the following modifications. Ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems. B hela cells were transfected with luciferase encoding plasmid dna using either transit.
The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. It is inactivated after in vivo gene transfer into the mouse liver and muscle. The reagent provides high transfection efficiency in many cells types and formats. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells.
The intracellular trafficking mechanism of lipofectaminebased. I use 24well plates for transfections in triplicates. 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. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. How does transfection with lipofectamine 2000 work. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. For using lipofectamine 2000 reagent, the above plasmids were mixed. Systematic screening of commonly used commercial transfection. Prewarm 50ml of optimum stored in cold room at 4c 10min. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Despite this, a satisfactory mechanismbased explanation of their.
Transient transfection of mammalian cells with lipofectamine. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lower confluence will achieve higher percentage of transfection but may yield a lower absolute number of transfected cells. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Transfection of exogenous gene in specific plasmids to various cells is. Invitrogen lipofectamine 2000 transfection reagent 15ml. Start any new transfection by testing the recommended four. Dna complexes were added to cells that had already been allowed to attach to the plate b. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. After 4h aspirate media and refeed cells with normal media. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. To test this hypothesis, we evaluated the te of lfn and dd on both.
Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. I want to try to use hela cell and lipofectamine 2000 to do. May 11, 2016 for te experiments, lipofectaminedna and dotapdopcdna complexes were prepared in optimem invitrogen by mixing for each well of 24well plates 0. I want to try to use hela cell and lipofectamine 2000 to. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines.
Genscript recommends using lipofectamine 2000 for all transfections. Higher densities can lead to spontaneous differentiation. 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. Knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. Do any one has protocol for stable transfection with lipofectamine 2000. Test serumfree media for compatibility with lipofectamine 2000 since some. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. 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. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Highest transfection efficiency in many cell types and formats e. The trusted, simple, broadspectrum transfection reagent for most cell lines. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. The media needs to be changed at least 1hr prior to transfection.