Lipofectamine 2000 transfection reagent from invitrogen. Plasmid measure concentration 293 t packaging cells at 1. 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. Guides for easy transfection of notsocom mon cell lines are also available at lipofectamine2000. Transfection and genome engineering fisher scientific. Invitrogen lipofectamine 2000 transfection reagent life. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. 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. Although invitrogen recommends their own brand of culture medium for transfection, called optimem, we found no difference between this and conventional media. Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i.
But unfortunately the survival rate of cells has only been 46,9 61,9 %. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. For additional information on scaling your transfection reaction, see page 4. Lipofectamine 2000 reagent thermo fisher scientific.
Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Highest transfection efficiency in many cell types and formats e. Mix1 different for each well the volumes given are per well. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Combine diluted dna and diluted cationic lipid reagent, mix, and.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Scaling up or down lipofectamine 3000 reagent transfections. Formulation of lipofectamine 2000 and lipofectin signagen blog. Cationic liposome and plasmid dna complexes formed in serum. Works effectively with all common cell lines as well as with many challenging ones, and can. Invitrogen transfection guide available at for a list of cell. The amount of lipofectamine 2000 should be changed in proportion to the culture medium. Invitrogen lipofectamine 2000 transfection reagent is a proprietary. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. However, recently i tried to combine overexpression with scratch wound healing assay. Transfection of human hepatoma hepg2 cells with metafectene pro. I wondering how much dna i should use for stable transfection with lipofectamine 2000 tranfection reagent.
Each tube gets 8mls of optimem, 32ug of gag pol, 12. Make dnalipofectamine 3000 complexes in serumfree medium such as. Lipofectamine 2000 reagent 3 l lipofectamine 2000 reagent 1. Lipofectamine 2000 is commonly used for sirna transfections. From the luciferase activities and consistencies of data using different transfection reagents including genejammer, effectine, wellfect, lipofectamine, what i can say from the data i observed, the efficiency of transfection is much more better in hct116 cells. How does transfection with lipofectamine 2000 work. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. Read 7 answers by scientists with 9 recommendations from their colleagues to the question asked by on oct, 2016. Lipofectamine 2000 from invitrogen biocompare product. I use 24well plates for transfections in triplicates. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Help frozen lipofectamine transfection and transduction.
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. The figure demonstrates that, similarly to lipofectamine 2000 which was employed at the concentration suggested by the manufacturer concentration of metafectene pro varying between 1,5 and 4,5 l caused little loss of viability 1020%. Regarding cytotoxicity of hct116 cells, they at confluency 70% or higher can tolerate of 1. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Do any one has protocol for stable transfection with lipofectamine 2000.
Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays. Lipofectamine 2000 is a proprietary formulation suitable for the transfection of. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Combine liposomes and dna by carefully pipetting the dna solution into the polystyrene tube. 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. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Lipofectamine reagents go online to view related products.
The negative log of the p values is the probability of. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Enable high transfection efficiency in novel genome editing applications. Invitrogen lipofectamine 3000 transfection reagent 0. Cationic liposomes are made of both cationic lipid and neutral phospholipid, conferring positively charged submicron particles, while plasmid dna is a hydrophilic molecule with a highly negative surface charge due to the presence of phosphate groups on. Product is guaranteed for 6 months from the date of shipment if. 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. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Prepare a mixture of the transfection plasmids with optimem 250ulwell. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells.
Effects of lipofectamine 2000sirna complexes on autophagy. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna. 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. Help on transfecting mcf7 and hct 116 cells transfection. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Effects of lipofectamine 2000sirna complexes on autophagy in.
Advanced transfection with lipofectamine 2000 reagent. Transient plasmid dna transfection into hela and hacat. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. However, few studies have examined cellular responses to this delivery system. Hi i am really worried, i transported my lipofectamine from one lab to another in a dry ice container, and when i arrived to my lab and i open the box i found that the lipofectamine has been frozen i put it immediatly at 4 c, but i dont know if i damaged the lipofectamine can i still use this lipofectamine, i need to do an adeasy transfection tomorrow and i dont know if is going to work. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. 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. Xinxin liu, updated august 2014 prsv 10 ug mix measure concentration pmdl 10 ug vsv 10 ug plenti. Prewarm 50ml of optimum stored in cold room at 4c 10min.
Help on transfecting mcf7 and hct 116 cells oct222008 hi all. Despite this, a satisfactory mechanismbased explanation of their. After 5 minute incubation, combine the diluted dna with diluted lipofectamine 2000 cd reagent total volume 100 l. Add 6l of lipofectamine2000 from biostores and 100 l of. Invitrogen lipofectamine 2000 transfection reagent 0. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Invitrogen lipofectamine 2000 transfection reagent fisher. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent. Transfection enhancing reagents such as plus reagent invitrogen may be added to.
Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Lipofectamine 2000 transfection reagent invitrogen yumpu. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The transfection protocols using lipofectamine 2000. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3.
Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. Effects of lipofectamine 2000 sirna complexes on autophagy in hepatoma cells. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. 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. What are the ways to troubleshoot transfection using. Cationic liposomemediated transfection lipofection has been used as an efficient method for direct gene transfer both in vitro and in vivo. Refer to the cell lines database for a list of cell types successfully. The intracellular trafficking mechanism of lipofectamine. Once the lipofectamine 2000 is diluted, combine it with the dna within 30 min.
Combine the two solutions, mix gently and incubate for 20 min. Lipofectamine is a cationic liposome formulation, which complexes with negatively charged nucleic acid molecules to allow them to overcome the electrostatic repulsion of the cell membrane. Ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems. Canonical pathway enrichment analysis shows the primary biological processes impacted in hela cells that were transfected with transit. The mechanism of lipofectamine 2000 mediated transmembrane. Transfection of cultured eukaryotic cells using cationic lipid. Download limit exceeded you have exceeded your daily download allowance. Whats the difference between lipofectine and lipofectamine.
Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold. Optimized conditions for successful transfection of human. 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. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells.
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