Lipofectamine contains lipid subunits that can form. Improved delivery of cas9 proteingrna complexes using. Lipofection is most widely used method for transfecting neurons for livecell imaging experiments. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. Lipofectamine 3000 reagent outperforms invitrogen lipofectamine 2000 and fugene hd reagents. Invitrogen lipofectamine 3000 transfection reagent from.
Highest transfection efficiency in many cell types and formats e. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Outline of protocol steps for the use of lipofectamine 2000 reagent. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. 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 reagents go online to view related products. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells.
Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i. Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. Descriptionlipofectamine 2000 is a proprietary formulation suitable for the transfection of. In this case, the cas9 rnps and lipofectamine crisprmax. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The optimal level of each transfection reagent was determined empirically and all reagents were used according to manufacturers protocol. Lipofectamine 2000 from invitrogen biocompare product. Photograph of expected results dna lipofectamine scaling up or down. How can i optimize the transfection efficiency using. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. Invitrogen lipofectamine 2000 transfection reagent life. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Protocol generally says using 12ug dna with 68ulwell lipofectamine in 6 well plates. Formulation of lipofectamine 2000 and lipofectin signagen blog.
Invitrogen lipofectamine rnaimax transfection reagent. Invitrogen lipofectamine 2000 transfection reagent 0. Optimization of sw480 colon cancer cells transfection with lipofectamine 2000. Lipofectamine 2000 is commonly used for sirna transfections. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results.
As a matter of fact, they should be evaluated in their entirety for the development of optimized nonviral gene delivery vectors. Lipofectaminea rnaimax reagent thermo fisher scientific. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold. Each reagent was used to transfect hek 293, hela, lncap, hepg2, and a549 cell lines in a 96well format, and gfp expression was analyzed 48 hours posttransfection. Advanced transfection with lipofectamine 2000 reagent. Transfections were performed in 24well plates using 0. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen geneart precision tals and crispr nucleases targeting the aavs1 safe harbor locus in u2os and hepg2 cell lines in 12well format. The intracellular trafficking mechanism of lipofectamine.
It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Being a lowefficiency method it results in very sparse distribution of transfected neurons, which is perfect for analysis of endosomes dynamics within single neurons. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 transfection reagent, invitrogen. Delivery of episomal vectors into primary cells by means of.
Product is guaranteed for 6 months from the date of shipment if. This study shows that lipofectamine 2000 is an efficient. Invitrogen lipofectamine 2000 transfection reagent 1. Product isguaranteed for 6 months from the date of shipment if stored properly. Dilute lipofectamine rnaimax reagent in optimem medium 2 dilute sirna in optimem medium 3 add diluted sirna to diluted lipofectamine rnaimax reagent 1. Lipofectamine 3000 reagent is suitable for novel genome editing applications. For additional information on scaling your transfection reaction, see page 4. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. Pdf optimization of sw480 colon cancer cells transfection with. Lipofectamine 2000 transfection protocol 10cm plates reagents. The mechanism of lipofectamine 2000 mediated transmembrane. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy.
Caco2 transfection reagent colon carcinoma cells altogen. Lipofectamine 2000 and lipofectamine rnaimax transfection. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Plasmid dna and lipofectamine 2000 invitrogen, carlsbad, ca were diluted in two independent 250.
Provides highest transfection efficiencies on widest variety of cell types for sirnamediated gene knockdown experiments. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Effects of lipofectamine 2000sirna complexes on autophagy in. 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 11668019 view more versions of this product. Add 6l of lipofectamine2000 from biostores and 100 l of. Sep, 2005 both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Invitrogen lipofectamine 2000 transfection reagent. Select the part name and then you can download the datasheet in pdf format. Invitrogen lipofectamine 3000 transfection reagent. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. 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. Invitrogen lipofectamine 3000 transfection reagent fisher.
It increases the likelihood of successful cleavage and recombination with talens or crisprs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes. Versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The trusted, simple, broadspectrum transfection reagent for most cell lines. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. I use 24well plates for transfections in triplicates.
This observation is consistent with reports that, regardless of the transfection protocol, delivery of. The inhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000 pgfpn2. This protocol is to produce lentivirus stocks from hairpinplko. Dilute your dna and lipofectamine mix in serum free medium. Transfections were conducted by the lipofectamine method. Lipofectamine 2000 is then added to culture medium within a sterile tube along with the dna at a 3. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Pea3 activates vegf transcription in t47d and skbr3 breast. 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. The purpose of this protocol is to introduce dna or small rna into eukaryotic cells for recombinant expression or down regulation of defined. Optimized conditions for successful transfection of human. 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. 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 dna lipofectamine 3000 complexes in serumfree medium such as.
Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Lipofectamine 2000 transfection reagent dartmouth college. The intracellular trafficking mechanism of lipofectaminebased. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. Lentiviral vectors for gene expression plasmids for transient and stable gene expression.
Contact life technologies technical service or your local sales representative for permission to crossreference the dmf. 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. Optimizing transfection lipofectamine 2000 cd reagent. In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. However, few studies have examined cellular responses to this delivery system. Lipofectamine 2000 dna transfection reagent protocol. 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.
Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Invitrogen lipofectamine 3000 transfection reagent 1. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. The ratio of transfection reagent to episomal vectors was varied, and the subsequent. Prewarm 50ml of optimum stored in cold room at 4c 10min. You can calculate this according to instructions in your reagent manual. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and.
Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Invitrogen lipofectamine 2000 transfection reagent strong performance in a wide range of cell lines reliableone of the mostcited dna reagents strong performancefor easytotransfect to moderately difficulttotransfect cell lines easy to usesimple protocol for easy optimization in a. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Invitrogen lipofectamine 2000 transfection reagent fisher. A reverse transfection protocol was used to transfect mcf7 and hepg2 cells. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Despite this, a satisfactory mechanismbased explanation of their.
Dilute sirnachimera with optimem i reduced serum medium invitrogen up to 50 l and vortex for a few seconds dilute appropriate amount of lipofectamine 2000 by optimem i reduced serum medium to 50 l and vortex for a few seconds for siha and skov3 cells, dilute 1. Invitrogen lipofectamine rnaimax transfection reagent 0. For lipofectamine plus transfections, the dna was preincubated with 4. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
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