Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. B hela cells were transfected with luciferase encoding plasmid dna using either transit. Prewarm 50ml of optimum stored in cold room at 4c 10min. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. 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. To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. The transfection efficacy of lipofectamine 2000 was compromised by its. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Lipofectamine 3000 protocol written out with timing. Is it possible to transfect cells with mrna less than 700bp using. With cell number and dna concentration held constant, vary the amount of lipofectamine 2000 to determine the optimal concentration usually 1. I want to try to use hela cell and lipofectamine 2000 to.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. 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. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Troubleshooting for transfection of the crisprcas9 using. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. So, i have these two plasmids which i need to transfect say x and y. In biontex experiments gfp positivity increased to 90. Use the procedure on below to transfect cells with plasmid dna. Invitrogen lipofectamine 3000 transfection reagent fisher. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Transient and stable transfection of adherent and suspension cells high throughput transfections delivery of stealth rnai and sirna into cells for information on transfecting mammalian cells with short interfering rnas sirna for use in rna interference rnai studies, visit our rnai central web page at \rnai. For additional information on scaling your transfection reaction, see page 4. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types.
The image below figure 1 is an example of the microarrays that can be created with this approach. Comparison of transfection efficiency of nonviral gene. Cotransfection of two plasmids need the protocol for lipofectamine 2000 based transfection nov 012009 hi, i am trying to cotransfect transient trasnfection two different plasmids in to hek293t cells. 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.
This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. It is known that lipidbased reagents tend to destabilise the plasma membrane, facilitating the smooth internalisation of lipoplexes 41. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. Systematic screening of commonly used commercial transfection. Highest transfection efficiency in many cell types and formats e.
Max % of positive cells after transfection of a gfpplasmid 3. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start with cells at 90% confluency and use a fixed amount of dna 0. Dilute dna in 250 l of optimem i reduced serum medium without serum or other medium without serum. Invitrogen lipofectamine 2000 transfection reagent fisher. 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 and suspension cell lines. If there appears to be largescale loss of cells after lipofectamine treatment, then it might pay to try using polylysinecoated plates. May 11, 2016 the intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. How does transfection with lipofectamine 2000 work. Lipofectamine 2000 and lipofectamine 3000 were used to transfect u2os and. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency.
Of noted, lipofectamine 2000 induced strong immune responses after transfection toward 293htlr9 as well as 293htlr7 cells, although responses in the former were lower than those in the latter. Invitrogen lipofectamine 3000 transfection reagent from. Transfection guide overview of transfection methods promega. 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. Cells should be plated on collagencoated dishes, as for normal propagation. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Comparison of three different transfection methods biology.
In vivo messenger rna introduction into the central. This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene delivery methods such as electroporation and viral infection. Scaling up or down lipofectamine 3000 reagent transfections. I have used both lipofectamine 2000 and 3000 and always get the same results. Lipofectamine 3000 protocol written out with timing specifics. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Jul 24, 2007 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. Electroporation was first reported for gene transfer studies in mouse cells. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection. Plate cells so they will be 7090% confluent at the time of transfection. We call this approach reverse transfection and we have developed two methods to reverse transfect cells. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. 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. Lipofectamine 2000 reagent thermo fisher scientific. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. In tube a place 2 g of dna in optimemi for a final volume of 200 l. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Cells were transfected with a firefly luciferase encoding plasmid dna 0. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Despite this, a satisfactory mechanismbased explanation of their. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Lipofectamine transfection of c2c12 cells bridges lab.
In this context, since their launch in 1993, lipofectamine lfn reagents have become the most used trs, with over. To transfect cells with sirna, follow the protocol as. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. I think you can use lipofectamine 3000 to transfect mrna. Refer to the cell lines database for a list of cell types successfully. To transfect neurons plated on two 15 mm coverslips each in one well of 12well plate or one 35 mm glassbottom dish as well as one 25 mm. Luciferase activity was measured after 24 hours figures 24. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic. 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. Jurkat cells appear to be less sensitive to toxicity than hek293 and hep g2 cells, as only slight decreases in viability were seen with all transfection reagents in these cells figure 4. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. An improved method for increasing the efficiency of gene. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Other thing is to incease the cell density to 8595% confluncy as suggested by the manufacturer.
Optimized conditions for successful transfection of human. From the above data in table 3, this indicates that the 1. Lipofectamine plasmid transfection bridges lab protocols. It is best to use polypropylene instead of polystyrene tubes. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids. Sep, 2005 lipofectamine 2000 is an easy to use transfection reagent. Lipofectamine2000 dna transfection reagent protocol transfect cells according to the following chart. The media needs to be changed at least 1hr prior to transfection. The transfection efficiencies of the pte cells were 5% effectene. I use 24well plates for transfections in triplicates.
Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start. Cotransfection of two plasmids tissue and cell culture. Maximal transfection efficiency in mc3t3e1, c3h10t12, hela, c2c12, hep g2, and hct116 was seen using fugene hd, in pt30, 4t1, and hekn was seen using arrestin, and in mcf7 was seen using jetpei. In a standard transfection protocol, the cells are plated on day 1, transfected on. Cells in a 6 well dish, plated and at 90% confluence.
The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetpei and expressfect. This protocol is for transfecting plasmid dna into mammalian cells. For suspension cells andor high throughput applications, a reverse transfection protocol where freshly passaged cells are added to preplated transfection complexes is ideal as it reduces handson time for the end user. 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.
I would like to transfect 293t cells with lipofectamine 3000. Anyone else have toxicity issues with lipofectamine 2000. The mechanism of lipofectamine 2000 mediated transmembrane. To transfect cells with sirna, follow the protocol as described for dna but. Lipofectamine 2000 transfection reagent was the most toxic, and produced as high as a 55% drop in viability in hek293 cells figure 2. Lipofectamine 2000 may be used in the following applications. I found this possibility in lipofectamine 2000 manual, but not in lipofectamine 3000 manual. Transfection was measured by luciferase activity using a conventional assay. Add 6l of lipofectamine2000 from biostores and 100 l of. You can calculate this according to instructions in your reagent manual. Hek293 cells and transduced huvecs compared with lipofectamine 2000 or. It is recommended that cells are growing at 9095% confluence prior to transfection for adherent cells. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
Invitrogen lipofectamine 3000 transfection reagent. Lipofectamine2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and mrna into a wide range of eukaryotic cells. Cells in a 6 well dish, plated and at 70% confluence. The vector contained an ofp reporter gene and was transfected. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. May 11, 2016 for te experiments, lipofectaminedna and dotapdopcdna complexes were prepared in optimem invitrogen by mixing for each well of 24well plates 0. Lipofectamine 2000 transfection reagent from invitrogen. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Transient plasmid dna transfection into hela and hacat cells. Versatileone reagent for dna, rna, and cotransfection. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. For each transfection sample, prepare dnalipofectamine 2000 complexes as follows. Nucleic acidlipofectamine2000 complexes can be added directly to cells in culture medium, in the presence or absence of serumantibiotic.
A sample protocol is listed here for transfection experiments performed in 6well plates. You can generally use other serumfree medium instead of optimem. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. 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. Enable high transfection efficiency in novel genome editing applications. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. A highly efficient method of transfecting hep g2 cells. Mix lipofectamine 2000 gently before use, then dilute the appropriate amount in 250.