Scaling up or down lipofectamine 3000 reagent transfections. Use the procedure on below to transfect cells with plasmid dna. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. This lipid reagent has proven to be effective for many different cultured mammalian cell lines. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. I have successfully transfected hct116 cells, but have been told ht29s are more difficult to transfect. Invitrogen lipofectamine 3000 transfection reagent l3000008. Lipofectamine 2000 right panel around 5% efficiency.
If you are using these cell lines or another mammalian cell line, we recommend optimizing transfection conditions to obtain the best results for your target gene and sirna. The highest transfection efficiency in many cell types and formats e. 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. Read advanced transfection with lipofectamine 2000 reagent. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Protocol for ht29 sirna transfection using lipofectamine 2000. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs.
Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Genscript recommends using lipofectamine 2000 for all transfections. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. A case study for optimization procedure jeanphilippe carralot, 1 taekyu kim, boris lenseigne,2 annette s. For optimal transfection conditions with the mission sirna transfection reagent, the for 1. 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. 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. We recommend gibco optimem i reduced serum medium cat. Use the procedure on page 3 to transfect cells with plasmid dna. Relaxing tea jazz elegant intrumental jazz music for work,study,reading relax music 4,195 watching live now. 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. Can i use lipofectamine 2000 to transfect with sirna. 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. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells.
This protocol is for transfecting plasmid dna into mammalian cells. The sirna transfection reagent is vided as a sterile solution, and is compatible with serum and antibiotics. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature. Afcs procedure protocol pp00000182 version 1, 103103. Using the transfection protocol outlined above, we routinely achieve transfection efficiencies of 5060% and get 5 to 20fold overexpression of protein. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Can anybody give me exact protocol for sirna transfection using rnaimax. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Ntersirna nanoparticlemediated knockdown of gene expression. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used. Transfection is the process of introducing nucleic acids into eukaryotic cells by nonviral methods. So i asked one of my lab mates and he told me to use just 1ul lipofectamine for 20pmol of sirna thats the protocol specificationsbut instead for each well, use that amount for three. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish.
Fast and efficient transfection of mouse embryonic stem. Lipofectamine 2000 reagent thermo fisher scientific. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best. The recommended cell density for transfection using lipofectaminetm 2000 is 9095%. I use 24well plates for transfections in triplicates. Transfection guide overview of transfection methods. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes.
Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Transfecting sirna into mammalian cells using lipofectaminez 2000, continued transfection procedure use the following procedure to transfect mammalian cells in a 24well format. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Mammalian cell penetration, sirna transfection, and dna. Automated highthroughput sirna transfection in raw 264. 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. Transfection protocol lipofectamine 3000 transfection. Optimized delivery of sirna into 3d tumor spheroid. In this video, we will perform an sirna transfection experiment using. Use the procedure on page 2 to transfect cells with short interfering rna sirna or invitrogen stealth rnai. Using lipofectaminez 2000 for transfection provides the following advantages. Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment. This protocol is well adjusted for cancer cell lines. Lipofectamine 2000 transfection reagent life technologies, cat.
Finally, we present a fast and efficient method for transfection of trypsinized mes cells using the liposomalbased lipofectamine 2000. Can anybody give me exact protocol for sirna transfection. Invitrogen lipofectamine 2000 transfection reagent. Mission sirna transfection reagent s1452 technical. Sep192012 hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. Rnai in cultured mammalian cells using synthetic sirnas. Transfection protocol transfection components component cat. 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. Protocol for ht29 sirna transfection using lipofectamine. Guidelines for transfection use the procedure on below to transfect cells with short interfering rna sirna or invitrogen stealth rnai. 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. All products were used according to manufacturers protocol. The protocal is from thermo fisher scientific important guidelines for transfection 1. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. B summary of increases in titer levels using two different cell lines and insert sizes. Cells in a 6 well dish, plated and at 70% confluence. Add 1 ul lipofectamine 2000 into 30 ul dmem without serum, mixing well and. 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. A comparison showing transfection efficiency of polyjet reagent vs. Lipofectamine 2000 and lipofectamine rnaimax transfection. Looking to do some preliminary experiments and i dont want to but a new reagent if i dont have to. Stealthsirna transfection protocol lipofectamine 2000. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. A titers obtained using lipofectamine 3000 reagent compared to commonly used transfection reagents in 293ft cells using a 3 kb insert gene.
Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. The development of reporter gene systems and selection methods for stable maintenance. 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. Lipofectamine transfection of c2c12 cells bridges lab. 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. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died.