However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used. Mammalian cell penetration, sirna transfection, and dna. Can anybody give me exact protocol for sirna transfection using rnaimax. Sep192012 hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. A comparison showing transfection efficiency of polyjet reagent vs. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Thermo scientific nunc 24well cellculture treated multidishes 142475 invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 step tube complexation components amount per well 24well. Transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best. This protocol is for transfecting plasmid dna into mammalian cells.
Relaxing tea jazz elegant intrumental jazz music for work,study,reading relax music 4,195 watching live now. 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 lipofectamine 3000 transfection reagent. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. I have successfully transfected hct116 cells, but have been told ht29s are more difficult to transfect.
Use the procedure on page 2 to transfect cells with short interfering rna sirna or invitrogen stealth rnai. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. 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. Protocol for ht29 sirna transfection using lipofectamine. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. In this video, we will perform an sirna transfection experiment using.
Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. The protocal is from thermo fisher scientific important guidelines for transfection 1. Cells in a 6 well dish, plated and at 70% confluence. Lipofectamine 2000 transfection reagent life technologies, cat. Rnai in cultured mammalian cells using synthetic sirnas. Transfection protocol transfection components component cat. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Fast and efficient transfection of mouse embryonic stem. The recommended cell density for transfection using lipofectaminetm 2000 is 9095%.
Guidelines for transfection use the procedure on below to transfect cells with short interfering rna sirna or invitrogen stealth rnai. Mission sirna transfection reagent s1452 technical. For optimal transfection conditions with the mission sirna transfection reagent, the for 1. I use 24well plates for transfections in triplicates. Boese,3 peter sommer,3 auguste genovesio,2 and priscille brodin1 rnai using sirna is a very powerful tool for functional genomics to identify new drug targets and biological pathways. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cellculture treated multidishes 142475 step tube complexation components amount per well 24well. Using the transfection protocol outlined above, we routinely achieve transfection efficiencies of 5060% and get 5 to 20fold overexpression of protein. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression.
Reverse transfection using ntersirna nanoparticles. Afcs procedure protocol pp00000182 version 1, 103103. In biomedical applications, this process of rna interference has encouraged researchers to study ways in which this process can be utilized to shut down or effectively incapacitate a defective or nonwanted genes ability to replicate. Scaling up or down lipofectamine 3000 reagent transfections. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Protocol for ht29 sirna transfection using lipofectamine 2000.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Add 1 ul lipofectamine 2000 into 30 ul dmem without serum, mixing well and. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Using lipofectaminez 2000 for transfection provides the following advantages. A titers obtained using lipofectamine 3000 reagent compared to commonly used transfection reagents in 293ft cells using a 3 kb insert gene. Use the procedure on below to transfect cells with plasmid dna. Invitrogen lipofectamine 2000 transfection reagent. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Finally, we present a fast and efficient method for transfection of trypsinized mes cells using the liposomalbased lipofectamine 2000. Stealthsirna transfection protocol lipofectamine 2000.
B summary of increases in titer levels using two different cell lines and insert sizes. A case study for optimization procedure jeanphilippe carralot, 1 taekyu kim, boris lenseigne,2 annette s. Optimized delivery of sirna into 3d tumor spheroid. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. Lipofectamine transfection of c2c12 cells bridges lab. The development of reporter gene systems and selection methods for stable maintenance. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature.
Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. L transfection of sirna to transfect cells with sirna, follow the protocol as described for dna but do not add p3000 reagent when diluting the sirna step 3. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. Lipofectamine 2000 and lipofectamine rnaimax transfection. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. If you wish to transfect cells in other tissue culture formats, you will need to determine the optimal conditions to use for your mammalian cell line. We recommend gibco optimem i reduced serum medium cat. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Invitrogen lipofectamine 3000 transfection reagent l3000008. Transfection protocol lipofectamine 3000 transfection. This lipid reagent has proven to be effective for many different cultured mammalian cell lines. Lipofectamine 2000 reagent thermo fisher scientific.
Can anybody give me exact protocol for sirna transfection. Read advanced transfection with lipofectamine 2000 reagent. 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. Lipofectamine 2000 right panel around 5% efficiency. Co transfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Automated highthroughput sirna transfection in raw 264. Transfection guide overview of transfection methods. Can i use lipofectamine 2000 to transfect with sirna. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Ntersirna nanoparticlemediated knockdown of gene expression.
The sirna transfection reagent is vided as a sterile solution, and is compatible with serum and antibiotics. Transfection protocols are used in biological laboratories for introduction of foreign plasmid dna, small therapeutic rna, or protein molecules into cells of a eukaryotic nature. Looking to do some preliminary experiments and i dont want to but a new reagent if i dont have to. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. In general, we recommend using lipofectaminetm 2000 or lipofectamintm plus from invitrogen. Use the procedure on page 3 to transfect cells with plasmid dna. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. A sample protocol is listed here for transfection experiments performed in 6well plates. This protocol is well adjusted for cancer cell lines. The highest transfection efficiency in many cell types and formats e.
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