Generally need to have 2 g of dna for each tranfection. Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. 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. 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. 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.
This protocol is for transfecting plasmid dna into mammalian cells. Rnai in cultured mammalian cells using synthetic sirnas ncbi. The protocal is from thermo fisher scientific important guidelines for transfection 1. 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. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. 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. 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. 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. Highest transfection efficiency in many cell types and formats e. 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.
I want to try to use hela cell and lipofectamine 2000 to do. This protocol can also be followed when cotransfecting plasmid dna and mirna by simply substituting mirna for sirna. Add the oligomerlipofectamine 2000 complexes from step 2. Stealthsirna transfection protocol lipofectamine 2000.
Hela transfection is usually pretty standard in terms of protocols just make sure your nucleic acid. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. We recommend gibco optimem i reduced serum medium cat. Use the procedure on below to transfect cells with plasmid dna. As always, use good cell culture practices and wear your personal protective equipment. This reference protocol provides general guidelines and procedure for plasmid dna and sirna reverse transfection into mammalian cells using magnetofectamine. Sep192012 hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. It may also refer to other methods and cell types, although other terms are often preferred. Use the procedure on page 3 to transfect cells with plasmid dna. 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.
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. 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. A general protocol for transfecting stealth rnai or sirna into mammalian cells can be. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Genscript recommends using lipofectamine 2000 for all transfections. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. The highest transfection efficiency in many cell types and formats e. Optimized delivery of sirna into 3d tumor spheroid cultures. Using lipofectaminez 2000 for transfection provides the following advantages. 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.
Transfecting sirna using lipofectamine rnaimax pin. Transfection guide overview of transfection methods. Step tube complexation components amount per well 24well. Genscript vectorbased sirna protocol technical manual no. Afcs procedure protocol pp00000182 version 1, 103103. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation. The complexes are prepared inside the wells, after which cells and medium are added. To transfect cells with sirna, follow the protocol as described for dna but. Transfection protocol lipofectamine 3000 transfection. Introduction a brief experienced users procedure for transfecting stealth rnai or sirna molecules into mammalian cells using lipofectamine 2000 reagent is described below. Rnai in cultured mammalian cells using synthetic sirnas. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used. How does transfection with lipofectamine 2000 work.
I use 24well plates for transfections in triplicates. Amounts of dna and reagent jetprime and competitors per well in 6well plate for transfection according to manufacturers recommendations. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Transfection protocol lipofectamine 3000 transfection reagent. Alternative transfection reagents can be used according to the manufacturers instructions, with this protocol serving as a. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Protocol for ht29 sirna transfection using lipofectamine. In animal cells, transfection is the preferred term as.
The transfection is designed for one rna amount combined with one amount of lipofectamine rnaimax. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting. Optimized conditions for successful transfection of human. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Transfection guide overview of transfection methods promega. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. I have successfully transfected hct116 cells, but have been told ht29s are more difficult to transfect. Optimizing plasmid dna transfections with lipofectamine 2000. The product manual includes advice on how to optimize transfection. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. 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. The sirna transfection reagent is vided as a sterile solution, and is compatible with serum and antibiotics. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Biomodule transfection and control user manual unit with.
Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Stealthsirna transfection protocol lipofectamine 2000 thermo. 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. 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. 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. This active endosome escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. Invitrogen lipofectamine 3000 transfection reagent l3000008. Some of my cells will dried if i used the volume suggested by the manual. Transfection efficiencies using commercially available reagents such as fugene hd and lipofectamine 2000 were generally low, but even the best. Comparison of small interfering rna sirna delivery into. Hi jasvinder, i recommend using a dedicated reagent for sirna transfection since. For optimal transfection conditions with the mission sirna transfection reagent, the for 1. 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. Guidelines for transfection use the procedure on below to transfect cells with short interfering rna sirna or invitrogen stealth rnai.
Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Lipofectamine transfection of c2c12 cells bridges lab. Optimemin ul, equal volume for dna and lipofectamine lipofectamine in ul 25. Knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna. Lipofectamine 2000 transfection reagent from invitrogen. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with. 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. Transfection was performed as described in protocol 2 with the following modifications. The recommended cell density for transfection using lipofectaminetm 2000 is 9095. The recommended cell density for transfection using lipofectaminetm 2000 is 9095%.
Lipofectamine 2000 transfection protocol 10cm plates reagents. 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. Too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Lipofectamine 2000 reagent thermo fisher scientific. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes.
Lipofectamine plasmid transfection bridges lab protocols. Comparative analysis of macrod1, macrod2 and targ1. Protocol for ht29 sirna transfection using lipofectamine 2000. Transfect plasmid dna and sirna at the same time using lipofectamine 2000. Lipofectamine 2000 is that it has an extensive, very useful manual. Mission sirna transfection reagent s1452 technical. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Use the procedure on page 2 to transfect cells with short interfering rna sirna or invitrogen stealth rnai. Step procedure preparing cells grow your mammalian cell line of choice in the recommended medium. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Cotransfection protocol for plasmid dna and sirnamirna. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Transfection efficiencies lower than 50% were not reported in this table.
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. Has anyone tried sirna transfection with lipofectamine3000. You will need to optimize the transfection for your purposes. Approximately 1824 hours before transfection, plate cells using the following guidelines. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. I would like to transfect 293t cells with lipofectamine 3000. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature. 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. Advanced transfection with lipofectamine 2000 reagent. Scaling up or down lipofectamine 3000 reagent transfections. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died.
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