After a 5 minute incubation in optimem, the lipofectamine 2000 and dna are combined and incubated for 20 minutes with gentle shaking at room temperature. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture. Despite this, a satisfactory mechanismbased explanation of their. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Jun 10, 2011 lipofectamine 2000 is commonly used for sirna transfections.
After 5 minutes incubation, combine the diluted dna with the diluted lipofectamine 2000 total volume is 500. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Schoenwolf1,2 we have used cationic lipidbased transfection reagents for ectopic gene expression experiments in developing vertebrate embryos. General procedure for transfection of mammalian cells with. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. The amount of lipofectamine 2000 should be changed in proportion to the culture medium. Thee two most important factors in choosing transfecting reagent are. Lipofectamine 2000 is supplied in liquid form at a concentration of 1 mgml. The intracellular trafficking mechanism of lipofectamine. Lipofectamine transfection of c2c12 cells bridges lab protocols. You can not use antibiotics during transfection step because it will cause cell death. I want to try to use hela cell and lipofectamine 2000 to do. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. But unfortunately the survival rate of cells has only been 46,9 61,9 %.
Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Lipofectamine transfection of c2c12 cells bridges lab. The optimal amount of dna to use in the transfection will vary widely depending upon the type of dna, transfection reagentmethod, target cell line, and number of cells genetic material dna quality and quantity factors affecting transfection 8. Transfection protocol for hpc7 using lipofectamine 2000 96 well. However, few studies have examined cellular responses to this delivery system. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. For each well to be transfected, prepare rnai duplex lipofectamine rnaimax complexes as follows. For additional information on scaling your transfection reaction, see page 4. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. For reverse transfection b, a549 cells were plated after adding complexes following the protocol below. Prewarm 50ml of optimum stored in cold room at 4c 10min. In biontex experiments gfp positivity increased to 90.
The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. I use 24well plates for transfections in triplicates. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. Optimized cationic lipidbased gene delivery reagents for use in developing vertebrate embryos michael decastro, 1yukio saijoh, and gary c. Lipofectamine 2000 dna transfection reagent protocol. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Lipofectamine 2000 is a proprietary formulation for the transfection of 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. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2. Product is guaranteed for 6 months from the date of shipment if stored properly.
Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Plate cells so they will be 6080% confluent at the time of transfection. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. A hela cells were transfected with hcv dsrna using lipofectamine 2000 reagent. Cells were transfected with a firefly luciferase encoding plasmid dna 0. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. Invitrogen lipofectamine 2000 transfection reagent life. Prepare 6 well plates by adding bakedsterile 22x22 1. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting. I am doing transfection using lipofectamine 2000 and plasmid dna, too. The reagent provides high transfection efficiency in many cells types and formats. 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.
To transfect cells with sirna, follow the protocol as described for dna but. After the 5 minute incubation, combine the diluted dna and rnai molecule with the diluted lipofectamine 2000. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Transient transfection of mammalian cells with lipofectamine. Considerations for optimal transfection using lipofectamine 2000 2. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Lipofectamine 2000 transfection reagent dartmouth college. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Combine the two solutions, mix gently and incubate for 20 min. Mar 03, 2014 to determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
Anyone else have toxicity issues with lipofectamine 2000. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. 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. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Maintain p19 cells undifferentiated in mem with 10% serum 7. What i found is lipofectamine 2000 is toxic to cells, so i reduced the recommended quantity of the invitrogen protocol. 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. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. C incubate at room temperature for 5min a combine the dna mixture with diluted lipofectamine 2000. The protocol indicated below is a reverse transfection protocol. After 5 minute incubation, combine the diluted dna. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure.
The microscopic analysis was performed using antirigi. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. Lipofectamine, lipofectamine 2000, and lipofectamine enhanced. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. This protocol is well adjusted for cancer cell lines. Each tube gets 8mls of optimem, 32ug of gag pol, 12. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000. Lipofectamine transient transfection of cos7 cells to. Lipofectamine 2000 transfection reagent from invitrogen. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Clean single portions keep the product fresh until use one step. I do have cell toxicity, but not so serious as yours. Lipofectamine 3000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to transfect cells important guidelines.
Higher densities can lead to spontaneous differentiation. Add 6l of lipofectamine2000 from biostores and 100 l of. After the 5 minute incubation, combine the diluted. Lipofectamine 2000 and lipofectamine rnaimax transfection.
Effects of lipofectamine 2000sirna complexes on autophagy in. Optimized cationic lipidbased gene delivery reagents for. Lipofectamine 2000 procedure for simultaneously transfecting. Lipofectamine 2000 reagent thermo fisher scientific. Genscript recommends using lipofectamine 2000 for all transfections. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website.
Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Advanced transfection with lipofectamine 2000 reagent. D after 20 min incubation a add the dnalipofectamine mix to the cells 1 mla 5 ml, the total medium will be 6 ml. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit.
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. In some cases, that accompanies with reduced transfection efficiency, sometimes it does not. Scaling up or down lipofectamine 3000 reagent transfections. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Rnai in cultured mammalian cells using synthetic sirnas. Pool dishes of cells that have been prepared for transfection. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Invitrogen lipofectamine 2000 transfection reagent fisher. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection.
Lipofectamine 2000 transfection protocol 10cm plates reagents. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Use the following procedure to transfect mammalian cells in a 24. 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. Transfection protocol 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 invitrogen lipofectamine 3000 transfection reagent. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. A sample protocol is listed here for transfection experiments performed in 6well plates.
To determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Plate cells so they will be 7090% confluent at the time of transfection. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable. It is possible to plate and transfect the cells on the same day, but. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Optimized conditions for successful transfection of human. This is most often an endogenous proteincoding gene, although some noncoding rnas can also be silenced effectively. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Transient plasmid dna transfection into hela and hacat cells.
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