Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. 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. However, few studies have examined cellular responses to this delivery system. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different. Studies were undertaken to evaluate and compare the transfection efficacy. Lipofectamine 2000 transfection reagent from invitrogen. Lipofectamine 3000 protocol written out with timing. Highest transfection efficiency in many cell types and formats e. General procedure for transfection of mammalian cells with.
How does transfection with lipofectamine 2000 work. I use 24well plates for transfections in triplicates. Lipofectamine 2000 is commonly used for sirna transfections. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. An example of high efficiency forward and reverse transfection of a549 cells using the broad spectrum transitx2 dynamic delivery system can be found in the figure below. Exceptional transfection efficiency in the broadest. If you can see successful transfection through a visual diagnostic e. To perform transfection experiments in other cell culture plates, simply multiply the. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from.
Transient plasmid dna transfection into hela and hacat cells. I have used both lipofectamine 2000 and 3000 and always get the same results. Rapid and highly efficient mammalian cell engineering via cas9 protein transfection. 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. 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 3000 protocol written out with timing specifics regarding the tran aug2016. Prepare plasmid dnalipid complexes recommend 2 doses of. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Rapid and highly efficient mammalian cell engineering via. 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. 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 2000 transfection reagent has been shown to effectively transfect the widest variety of adherent and suspension cell lines.
Lipofectamine 2000 transfection protocol 10cm plates reagents. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Numerous chemical and physical approaches have been used to deliver the dna, but their efficacy has been variable and is highly dependent on the cell type to be transfected. The lenti293t cell line has been tested as a suitable cell line for lentiviral production. May 11, 2016 for te experiments, lipofectaminedna and dotapdopcdna complexes were prepared in optimem invitrogen by mixing for each well of 24well plates 0. Genscript recommends using lipofectamine 2000 for all transfections. Troubleshooting for transfection of the crisprcas9 using lipofectamine. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone has found an optimal lipo 3000 dna ratio for this cell line. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy.
The intracellular trafficking mechanism of lipofectamine. Transfection protocol of lipofectamine rnaimax can be. What is the major character of lipofectamine 2000 as a. In tube a place 2 g of dna in optimemi for a final volume of 200 l. A sample protocol is listed here for transfection experiments performed in 6well plates. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Lipofectamine 2000 reagent thermo fisher scientific. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. A highly efficient method of transfecting hep g2 cells. To identify the best transfection reagents for delivery of cas9 rnps, initially we compared several commercially available protein transfection reagents, including lipofectamine 2000. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.
Stealthsirna transfection protocol lipofectamine 2000. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Each tube gets 8mls of optimem, 32ug of gag pol, 12. Transient plasmid dna transfection into hela and hacat. I want to try to use hela cell and lipofectamine 2000 to. What percentage confluency should be used for stable transfection. This protocol is for transfecting plasmid dna into mammalian cells. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Plate cells for 212hrs but not over 24 hours prior to transfection to. 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. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1.
The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. 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 lenti293t is an hek 293 derived cell line stably expresses the sv40 large t antigen and neomycin resistance gene. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited.
Plate cells so they will be 7090% confluent at the time of transfection. Optimized conditions for successful transfection of human. I am about to start my stable transfections with lipofectamine 2000. The mechanism of lipofectamine 2000 mediated transmembrane. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent. Comparative transfection of dna into primary and transformed mammalian cells from different lineages.
Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Using lipofectaminez 2000 for transfection provides the following advantages. What is the major character of lipofectamine 2000 as a transfection reagent. Lipofectamine 2000 is being used as a transfection reagent in vitro, what is the difference between this carrier and. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. 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. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Improved delivery of cas9 proteingrna complexes using. Version 1, 41 5 g pipet up and down 1215 times with a 10 ml pipet until a single cell suspension is obtained. The reagent provides high transfection efficiency in many cells types and formats. Superior performance for co transfection of sirna and plasmid dna. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Invitrogen lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Cotransfection of two plasmids need the protocol for lipofectamine 2000 based transfection. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Lipofectamine is formulated to give high transfection efficiency with little. I am using lipofectamine 2000 with optimem to transfect hek cells, but i havent had much luck so far. I normally incubate my cells in media with antibiotics and serum, and a. The highest transfection efficiency in many cell types and formats e. Prewarm 50ml of optimum stored in cold room at 4c 10min. The delivery of dna into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies.
Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Invitrogen lipofectamine 3000 transfection reagent. Do any one has protocol for stable transfection with lipofectamine 2000. Scaling up or down lipofectamine 3000 reagent transfections. Troubleshooting for transfection of the crisprcas9 using. Lipofectamine transfection of c2c12 cells bridges lab. I want to try to use hela cell and lipofectamine 2000 to do transfection. Refer to the cell lines database for a list of cell types successfully transfected. Add 6l of lipofectamine2000 from biostores and 100 l of. Lipofectamine 2000 transfection reagent dartmouth college.
Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. My problem is i am using lipofectamine have to use this as no other transfection reagents available to me to transfcet my cells today with a luciferase reporter gene the cells i am transfecting today are cells which were immortalised a few months back with psv3neo which has a selection marker for neomycin. It is used to increase the transfection efficiency of rna. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Cotransfection of two plasmids tissue and cell culture.
Luciferase activity was measured after 24 hours figures 24. Effects of lipofectamine 2000sirna complexes on autophagy in. Important guidelines for transfection use optimem i reduced serum medium cat. What are the ways to troubleshoot transfection using lipofectamine 3000 reagent and. Invitrogen lipofectamine 2000 transfection reagent fisher. Transient transfection of mammalian cells with lipofectamine. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number.
Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The use of these different lipofectamine 2000 amounts for transfection showed. To test the transfection efficiency of the reverse transfection approach, a luciferaseencoding plasmid was reverse transfected into hek293, hep g2 and jurkat cells using fugene 6, fugene hd, transitlt1 and lipofectamine 2000 transfection reagents. 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.