Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Jul 24, 2007 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. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Hela transfection is usually pretty standard in terms of protocols just make sure your nucleic acid. A sample protocol is listed here for transfection experiments performed in 6well plates. Genscript vectorbased sirna protocol technical manual no. Optimized delivery of sirna into 3d tumor spheroid cultures. To transfect cells with sirna, follow the protocol as described for dna but. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. 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.
Transfection was performed as described in protocol 2 with the following modifications. 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. 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 2000 is that it has an extensive, very useful manual. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Biomodule transfection and control user manual unit with. Use the procedure on below to transfect cells with plasmid dna. It may also refer to other methods and cell types, although other terms are often preferred. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. This reference protocol provides general guidelines and procedure for plasmid dna and sirna reverse transfection into mammalian cells using magnetofectamine. The transfection is designed for one rna amount combined with one amount of lipofectamine rnaimax. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Step tube complexation components amount per well 24well.
This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. Stealthsirna transfection protocol lipofectamine 2000. Advanced transfection with lipofectamine 2000 reagent. Transfection protocol lipofectamine 3000 transfection. Transfect plasmid dna and sirna at the same time using lipofectamine 2000.
Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation. A general protocol for transfecting stealth rnai or sirna into mammalian cells can be. 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. 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. Sep192012 hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. Therefore, all three transfection reagents appear suitable for use in bmdm. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for some cell lines where other lipidbased reagents result in lower transfection efficiencies table 1. Scaling up or down lipofectamine 3000 reagent transfections. Approximately 1824 hours before transfection, plate cells using the following guidelines. Has anyone tried sirna transfection with lipofectamine3000. Amongst the eleven transfection reagents investigated, lipofectamine 2000, lipofectamine rnaimax and dharmafect 3 consistently gave the best results, in terms of sirna uptake and target gene knockdown with minimal cell toxicity and type i ifn response induction. Optimizing plasmid dna transfections with lipofectamine 2000.
Comparison of small interfering rna sirna delivery into. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. We recommend gibco optimem i reduced serum medium cat. Reverse transfection for suspension cells andor high throughput applications, a reverse transfection protocol where freshly passaged cells are added to preplated transfection complexes is ideal as it reduces handson time for the end user. Use the procedure on page 3 to transfect cells with plasmid dna. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0.
I would recommend however, that you get some lipofectamine rnaimax instead of 2000, it is a lot less toxic and seems to work really well for sirna transfection. I am performing transfection on mcf7 cells using lipofectamine 3000 invitrogen but unable to get any transfectants, instead the cells are dying after incubation with reagent and dna. Add the oligomerlipofectamine 2000 complexes from step 2. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Transfection efficiencies lower than 50% were not reported in this table. Stealthsirna transfection protocol lipofectamine 2000 thermo. Genscript recommends using lipofectamine 2000 for all transfections. Generally need to have 2 g of dna for each tranfection. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. 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 wells. In animal cells, transfection is the preferred term as.
Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting. Lipofectamine transfection of c2c12 cells bridges lab. How does transfection with lipofectamine 2000 work. Protocol 6 well dish plate cells the day before so that they are at 9095% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Introduction a brief experienced users procedure for transfecting stealth rnai or sirna molecules into mammalian cells using lipofectamine 2000 reagent is described below. Transfection guide overview of transfection methods. Apr 15, 2014 amongst the eleven transfection reagents investigated, lipofectamine 2000, lipofectamine rnaimax and dharmafect 3 consistently gave the best results, in terms of sirna uptake and target gene knockdown with minimal cell toxicity and type i ifn response induction. Step procedure preparing cells grow your mammalian cell line of choice in the recommended medium. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. Afcs procedure protocol pp00000182 version 1, 103103. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
As always, use good cell culture practices and wear your personal protective equipment. We use cookies to offer you a better experience, personalize content, tailor advertising, provide social media features, and better understand the use of our services. 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. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Comparative analysis of macrod1, macrod2 and targ1. Highest transfection efficiency in many cell types and formats e. 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. This protocol is for transfecting plasmid dna into mammalian cells. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with. The product manual includes advice on how to optimize transfection.
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. Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Protocol for ht29 sirna transfection using lipofectamine. Transfecting sirna using lipofectamine rnaimax pin. Rnai in cultured mammalian cells using synthetic sirnas ncbi. Guidelines for transfection use the procedure on below to transfect cells with short interfering rna sirna or invitrogen stealth rnai. The recommended cell density for transfection using lipofectaminetm 2000 is 9095.
Lipofectamine 2000 reagent thermo fisher scientific. This protocol can also be followed when cotransfecting plasmid dna and mirna by simply substituting mirna for sirna. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna. I want to try to use hela cell and lipofectamine 2000 to do. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. For the analysis of mitochondrial morphology upon sirna transfection cells were transfected using lipofectamine rnaimax according to the manual for 48 hours prior to fixation.
Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. 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. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. As always, use good cell culture practices and wear your personal protective.
I would like to transfect 293t cells with lipofectamine 3000. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Optimized conditions for successful transfection of human. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature. You will need to optimize the transfection for your purposes. I have successfully transfected hct116 cells, but have been told ht29s are more difficult to transfect. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection.
May 21, 2018 transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best performing polymeric nanoparticles transfected 57. Cotransfection protocol for plasmid dna and sirnamirna. Amounts of dna and reagent jetprime and competitors per well in 6well plate for transfection according to manufacturers recommendations. The highest transfection efficiency in many cell types and formats e. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. A typical forward transfection protocol using transit2020 transfection reagent can be found here. Mission sirna transfection reagent s1452 technical. Transfection guide overview of transfection methods promega. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. Some of my cells will dried if i used the volume suggested by the manual. Transfection protocol lipofectamine 3000 transfection reagent. The complexes are prepared inside the wells, after which cells and medium are added. 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. The recommended cell density for transfection using lipofectaminetm 2000 is 9095%. This active endosome escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. Lipofectamine 2000 transfection reagent from invitrogen. For optimal transfection conditions with the mission sirna transfection reagent, the for 1. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection.
Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. 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. Alternative transfection reagents can be used according to the manufacturers instructions, with this protocol serving as a. Invitrogen lipofectamine 3000 transfection reagent l3000008. Protocol for ht29 sirna transfection using lipofectamine 2000. Lipofectamine plasmid transfection bridges lab protocols.