Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Transfection description materials optimum in fridge, dna in freezer, lipofectamine in fridge. Invitrogen lipofectamine 3000 transfection reagent. I attached the protocol which we use for invitrogen l2000. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg.
Does anyone have experience with the lipofectamine 2000 toxicity. Lipofectamine 3000 transfection reagent hepg2 liver. The average lentiviral titers in our preparations are around 56x 10 5 x 10. We tend to follow the protocol provided, which is simple and easy to follow. The mechanism of lipofectamine 2000 mediated transmembrane. Being a lowefficiency method it results in very sparse distribution of transfected neurons, which is perfect for analysis of endosomes dynamics within single neurons. Comparison of hsv, adenovirus, aav, retrovirus and lentivirus for gene delivery. The protocal is from thermo fisher scientific important guidelines for transfection 1. Transfection protocol transfection components component cat. Lipofectamine 2000 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Invitrogen lipofectamine 2000 transfection reagent. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to transfect 17 cell lines with a gfpexpressing plasmid in a 24well plate format using 0.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Optimem reduced serum medium, no phenol red life technologies, cat. Comparison of k2 transfection system with lipofectamine 2000 invitrogen for the transient transfection of hek293t cells. At 30 to 48 hours after the addition of dna, viruscontaining supernatant was harvested for p24 elisa, or. 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. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology.
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. I am performing transfection on mcf7 cells using lipofectamine 3000 invitrogen but unable to get any transfectants, instead. Lipofection is most widely used method for transfecting neurons for livecell imaging experiments. Add 6l of lipofectamine2000 from biostores and 100 l of. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. Lipofectamine 2000 dna transfection reagent protocol. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. 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. Optimized conditions for successful transfection of human. In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway were investigated. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Lipofectamine 2000 invitrogen or polyethylenimine pei transfection reagent sigmaaldrich. The intracellular trafficking mechanism of lipofectamine based transfection reagents and its implication for gene delivery. The neuronal cytoskeleton, motor proteins, and organelle trafficking in the axon.
Dec 01, 2006 invitrogens lipofectamine 2000 is highly cited in the literature, a sign of a successful reagent, so when our lab embarked upon the path to develop a successful transfection protocol, we included lipofectamine 2000 along with the other leading reagents. Msc victor van puyenbroeck, rega institute for medical research. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l lipofectamine 3000 reagent 1. Lipofectamine 2000 from invitrogen biocompare product. Evaluation of transfection methods for transient gene. It is used to increase the transfection efficiency of. Transfection and genome engineering fisher scientific. Researchers use lipofectamine 2000 reagent for plasmid dna transfection as well as sirna and shrnabased gene knockdown. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. Lipofectamine 2000 reagent thermo fisher scientific. Life technologies corporation andor its affiliates warrant their. The inhibitors of endocytosis were used to treat hep2 cells before lipofectamine 2000 pgfpn2. Transfection protocol lipofectamine 3000 transfection reagent.
Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Whats the difference between lipofectine and lipofectamine. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. This study shows that lipofectamine 2000 is an efficient reagent for the delivery of genes into sw480 cells. Works effectively with all common cell lines as well as with many challenging ones, and can. Photograph of expected results dna lipofectamine scaling up or down. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. 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.
Plasmid dna and lipofectamine 2000 invitrogen, carlsbad, ca were diluted in two independent 250. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. Lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine reagents go online to view related products. 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. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. 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 2000 transfection reagent 1.
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, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin. Transfection efficiency in a cancer cell line panel. The diluted dna and lipofectamine 2000 were combined and incubated at. Transfection of mcf7 with lipofectamine 2000 maartje vogel.
Prewarm 50ml of optimum stored in cold room at 4c 10min. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well. Product isguaranteed for 6 months from the date of shipment if stored properly. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. I want to try to use hela cell and lipofectamine 2000 to do. Lipofectamine 2000 and lipofectamine 3000 reagents deliver dna or cotransfect with excellent transfection performance for protein expression, gene silencing, and functional assays. Lipofectamine 2000, 3000, messengermax, and rnaimax. 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 is a proprietary. Invitrogen lipofectamine 3000 transfection reagent fisher.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Clathrin and caveolaemediated endocytic pathway contributions to transfection efficiency were studied. Lipofectamine 2000 reagent and lipofectamine ltx reagent. Invitrogen s lipofectamine 2000 is highly cited in the literature, a sign of a successful reagent, so when our lab embarked upon the path to develop a successful transfection protocol, we included lipofectamine 2000. 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. Can anyone provide me with a proper transfection protocol into. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Lipofectamine 2000 transfection reagent is a proprietary formulation. 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. Enable high transfection efficiency in novel genome editing applications. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2.
Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Invitrogen lipofectamine 3000 transfection reagent 1. The lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna, and lipofectamine messengermax reagent delivers mrna. Maintain p19 cells undifferentiated in mem with 10% serum 7. Comparison of k2 transfection system with lipofectamine. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number.
Make dnalipofectamine 3000 complexes in serumfree medium such as. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent. Pdf optimization of sw480 colon cancer cells transfection. Lipofectamine 2000 transfection protocol 10cm plates 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. The intracellular trafficking mechanism of lipofectamine. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Supplementary information for psgl 1 restricts hiv 1. Invitrogen lipofectamine 2000 transfection reagent fisher. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. 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. Lfn 2000 transfection reagent life technologies, carlsbad. I use 24well plates for transfections in triplicates. Lipofectamine 2000 transfection reagent from invitrogen.
Lipofectamine transfection of c2c12 cells bridges lab. Highest transfection efficiency in many cell types and formats e. Formulation of lipofectamine 2000 and lipofectin signagen blog. Lipofectamine 2000 transfection reagent life technologies, cat. I can say with confidence that invitrogen hela based chart never really worked out for me. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine. Lipofectamine 2000 transfection reagent from thermo fisher. Lipofectamine 2000 and lipofectamine rnaimax transfection. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 is a highperformance transfection reagent for gene expression and gene silencing. Does anyone have experience with the lipofectamine 2000.
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