Improved delivery of cas9 proteingrna complexes using. 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 2000 and lipofectamine rnaimax transfection. Lipofectamine 2000 reagent thermo fisher scientific. Lentiviral vectors for gene expression plasmids for transient and stable gene expression.
It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. This study shows that lipofectamine 2000 is an efficient. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The intracellular trafficking mechanism of lipofectamine. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. 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 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 is then added to culture medium within a sterile tube along with the dna at a 3. 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. Lipofection is most widely used method for transfecting neurons for livecell imaging experiments. Invitrogen 11668019 view more versions of this product. Caco2 transfection reagent colon carcinoma cells altogen.
Make dna lipofectamine 3000 complexes in serumfree medium such as. As a matter of fact, they should be evaluated in their entirety for the development of optimized nonviral gene delivery vectors. Lipofectamine 2000 dna transfection reagent protocol. Photograph of expected results dna lipofectamine scaling up or down. 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. 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. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. 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. Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. Descriptionlipofectamine 2000 is a proprietary formulation suitable for the transfection of. Dilute lipofectamine rnaimax reagent in optimem medium 2 dilute sirna in optimem medium 3 add diluted sirna to diluted lipofectamine rnaimax reagent 1.
Transfections were conducted by the lipofectamine method. Invitrogen lipofectamine rnaimax transfection reagent. Add 6l of lipofectamine2000 from biostores and 100 l of. 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. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Product is guaranteed for 6 months from the date of shipment if stored properly.
The mechanism of lipofectamine 2000 mediated transmembrane. Lipofectamine contains lipid subunits that can form. Lipofectamine 3000 reagent is suitable for novel genome editing applications. The trusted, simple, broadspectrum transfection reagent for most cell lines. Dilute your dna and lipofectamine mix in serum free medium. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. How can i optimize the transfection efficiency using. 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. Optimizing transfection lipofectamine 2000 cd reagent. Being a lowefficiency method it results in very sparse distribution of transfected neurons, which is perfect for analysis of endosomes dynamics within single neurons. 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. Optimization of sw480 colon cancer cells transfection with lipofectamine 2000. Invitrogen lipofectamine 3000 transfection reagent 1. Invitrogen lipofectamine 2000 transfection reagent 1.
Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i. Invitrogen lipofectamine 2000 transfection reagent 0. Plasmid dna and lipofectamine 2000 invitrogen, carlsbad, ca were diluted in two independent 250.
Invitrogen lipofectamine 3000 transfection reagent fisher. Highest transfection efficiency in many cell types and formats e. Lipofectaminea rnaimax reagent thermo fisher scientific. Product isguaranteed for 6 months from the date of shipment if stored properly. In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. 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.
Invitrogen lipofectamine 3000 transfection reagent from. The intracellular trafficking mechanism of lipofectaminebased. Despite this, a satisfactory mechanismbased explanation of their. Invitrogen lipofectamine 2000 transfection reagent.
However, few studies have examined cellular responses to this delivery system. Invitrogen lipofectamine 3000 transfection reagent. 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. The ratio of transfection reagent to episomal vectors was varied, and the subsequent. Invitrogen lipofectamine rnaimax transfection reagent 0. For lipofectamine plus transfections, the dna was preincubated with 4. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Lipofectamine 2000 transfection reagent, invitrogen. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. You can calculate this according to instructions in your reagent manual. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Provides highest transfection efficiencies on widest variety of cell types for sirnamediated gene knockdown experiments.
The optimal level of each transfection reagent was determined empirically and all reagents were used according to manufacturers protocol. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. For additional information on scaling your transfection reaction, see page 4. 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. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. A reverse transfection protocol was used to transfect mcf7 and hepg2 cells. This protocol is to produce lentivirus stocks from hairpinplko. Advanced transfection with lipofectamine 2000 reagent. Select the part name and then you can download the datasheet in pdf format. Versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. 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.
Protocol generally says using 12ug dna with 68ulwell lipofectamine in 6 well plates. Lipofectamine 2000 transfection protocol 10cm plates reagents. Prewarm 50ml of optimum stored in cold room at 4c 10min. Lipofectamine 2000 transfection reagent dartmouth college. Invitrogen lipofectamine 2000 transfection reagent fisher. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Pdf optimization of sw480 colon cancer cells transfection. Product is guaranteed for 6 months from the date of shipment if. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. The purpose of this protocol is to introduce dna or small rna into eukaryotic cells for recombinant expression or down regulation of defined. Lipofectamine 2000 is commonly used for sirna transfections. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
Transfections were performed in 24well plates using 0. This observation is consistent with reports that, regardless of the transfection protocol, delivery of. Pdf optimization of sw480 colon cancer cells transfection with. The inhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000 pgfpn2. 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. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. 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. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Pea3 activates vegf transcription in t47d and skbr3 breast.
Delivery of episomal vectors into primary cells by means of. Lipofectamine reagents go online to view related products. Although invitrogen recommends their own brand of culture medium for transfection, called optimem, we found no difference between this and conventional media. In this case, the cas9 rnps and lipofectamine crisprmax. Outline of protocol steps for the use of lipofectamine 2000 reagent. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Mar 03, 2014 to determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Effects of lipofectamine 2000sirna complexes on autophagy in. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines.
I use 24well plates for transfections in triplicates. Lipofectamine 2000 from invitrogen biocompare product. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Optimized conditions for successful transfection of human. Contact life technologies technical service or your local sales representative for permission to crossreference the dmf. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 3000 reagent outperforms invitrogen lipofectamine 2000 and fugene hd reagents.