Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Luciferase activity was measured after 24 hours figures 24. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. The day before transfection, pass confluent cell 1. 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. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Transfection description materials optimum in fridge, dna in freezer, lipofectamine in fridge.
Rnaimax reverse transfections lipofectamine thermo fisher. Prewarm 50ml of optimum stored in cold room at 4c 10min. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. However, whilst reverse genetics methods have been usually applied to. Akin to forward transfection, optimizing transfection conditions by transfecting a reporter plasmid into the cell type of interest such as a luciferase or gfp encoding plasmid into cells is critical prior to high throughout screening. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Arpe19 transfection kit retinal pigment epithelium. Lipofectamine 3000 reagent outperforms lipofectamine 2000 and. Here, we describe a robust protocol for the reverse transfection of cells on small interfering sirna arrays, which, in combination with multichannel immunofluorescence or timelapse microscopy. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells.
Genscript recommends using lipofectamine 2000 for all transfections. In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagentdna mix and. Our lab uses lipofectamine 3000 for transfections, but the only protocols for reverse transfection i can find on thermofisher scientifics website deal with their rnaimax reagent. The protocol given here is modified from a previously published protocol used for solidphase transfection in a 96well format erfle et al.
In short, a different rnai molecule is put in each well prior to transfection and combined with diluted lipofectamine 2000 to form complexes in each well. In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. The transfected cell microarray forms in 12 days and is then ready for downstream assays. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Maintain p19 cells undifferentiated in mem with 10% serum 7. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Transfection guide overview of transfection methods promega. Has anyone here attempted reverse transfection using. Reverse transfection combines rna, transfection reagent, and cells in an altered sequence compared to traditional lipofectamine 2000 transfection protocols. To test the transfection efficiency of the reverse transfection approach, a luciferaseencoding plasmid was reverse transfected into hek293, hep g2 and jurkat cells using fugene 6, fugene hd, transitlt1 and lipofectamine 2000 transfection reagents. Lipofectamine transfection of c2c12 cells bridges lab. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Version 1, 41 8 during the incubation of the printed slides with the transfection reagent, harvest hek293t cells and suspend 10 x 106 cells in 25 ml medium dmem with 10% ifs, 50 unitsml penicillin and 50 gml streptomycin.
Reverse transfection of stealth rnai or sirna using. 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. Application protocol reverse transfection of sirna on eplates. Both forward and reverse transfection using transitx2 dynamic delivery system yield superior luciferase expression compared to lipofectamine 2000. Application note gene delivery solutions stem cell. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. When performing a transient transfection, you can choose between a standard or reverse transfection protocol. Use this procedure to reverse transfect stealth rnai or sirna into mammalian cells in a 24well. 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. We tested multiple transfection reagents including lipofectamine. A typical forward transfection protocol using transit2020 transfection reagent can be found here pdf. Can anyone provide me with a proper transfection protocol. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart.
L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Similarly, accuracy and reproducibility in preparing the plates for solidphase reverse transfection of sirnas are also critical. For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Reverse transfection is a technique for the transfer of genetic material into cells. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes.
As dna is printed on a glass slide for the transfection process the deliberate introduction of nucleic acids into cells to occur before the addition of adherent cells, the order of addition of dna and adherent cells is reverse that of conventional transfection. New reverse genetics and transfection methods to rescue. Add 6l of lipofectamine2000 from biostores and 100 l of. In a reverse transfection protocol, cells are added directly to a plate. Transfection protocol lipofectamine 3000 transfection reagent.
Higher densities can lead to spontaneous differentiation. For reverse transfection, cells in optimal growth phase are prepared so that it is possible to. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. After removal of the transfection reagent, the slide is placed in a culture dish and covered with cells in media. For additional information on scaling your transfection reaction, see page 4. Mirus bio llc and lipofectamine 2000 transfection reagent life technologies. We have found that effectene, lipofectamine 2000 and fugene 6 work. Reverse transfection an overview sciencedirect topics.
For your convenience, the essential components of this protocol are now available in the gibco breast cancer starter kit. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Print bookmark share the next generation in lipid technology pdf. Before you start akin to forward transfection, optimizing transfection conditions by transfecting a reporter plasmid into the cell type of interest such as a luciferase or gfp encoding plasmid. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent.
Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Transfecting sirna using lipofectamine rnaimax pin liu, 2017. Transfection and genome engineering fisher scientific. Do not add antibiotics to media during transfection as this causes cell death. Our lab uses lipofectamine 3000 for transfections, but the only protocols for reverse. I use 24well plates for transfections in triplicates. Lipofectamine 2000 right panel around 5% efficiency. Invitrogen lipofectamine 3000 transfection reagent cat. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. This protocol is based on that from life technologies called. Here, we describe a robust protocol for the reverse transfection of cells on small interfering sirna arrays, which, in combination with multichannel immunofluorescence or.
For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. A comparison showing transfection efficiency of polyjet reagent vs. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. I tried reversetransfecting hepg2 cells using lipofectamine 2000. Protocol outline for transfection of pluripotent stem cells with lipofectamine 3000 reagent. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Reverse transfection protocol acea biosciences inc. Reverse transfection on cell arrays for high content. A sample protocol is listed here for transfection experiments performed in 6well plates. Forward and reverse transfection protocols mirus bio. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. The method illustrated is the gelatindna method of the reverse transfection approach.
Reverse transfection of stealth rnai or sirna using lipofectamine. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Lipofectamine 2000 transfection protocol 10cm plates reagents. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Using 384well plates has the advantage of significantly reducing.
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