Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. A lipidbased transfection reagent, such as xtremegene sirna transfection reagent, can provide a convenient, reliable, and efficient vehicle for delivering sirnas into animal cells, enabling the study of cellular and functional consequences of gene knockdown. Biomodule transfection and control user manual unit with. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. Optimized delivery of sirna into 3d tumor spheroid cultures. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine 3000 reagent sirna lipofectamine 3000 reagent 96well 0. Transfection protocol lipofectamine 3000 transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell. 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. Prepare plasmid dnalipid complexes recommend 2 doses of lipid.
Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. 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. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. To transfect cells with sirna, follow the protocol as. Highest transfection efficiency in many cell types and formats e.
Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. The vector contained an ofp reporter gene and was transfected. General description short interfering rna sirna can be directly introduced into cells by transfection. Step procedure preparing cells grow your mammalian cell line of choice in the recommended medium. Add the oligomerlipofectamine 2000 complexes from step 2.
Genscript recommends using lipofectamine 2000 for all transfections. Stealthsirna transfection protocol lipofectamine 2000. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature. After the 5min incubation, combine the diluted sirna with the diluted lipofectamine 2000 total volume is 100. I want to try to use hela cell and lipofectamine 2000 to. Add 6l of lipofectamine2000 from biostores and 100 l of. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used.
For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Stealthsirna transfection protocol lipofectamine 2000 thermo. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Scaling up or down lipofectamine 3000 reagent transfections. Has anyone tried sirna transfection with lipofectamine3000. Introduction a brief experienced users procedure for transfecting stealth rnai or sirna molecules into mammalian cells using lipofectamine 2000 reagent is described below. Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent. Reliable and efficient dna and rna transfection methods are required.
For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using lipofectamine. Invitrogen lipofectamine 2000 transfection reagent. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. To transfect cells with sirna, follow the protocol as described for dna but. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Fast and efficient transfection of mouse embryonic stem cells. Although this amount is good for a wide range of sirna concentrations, if more sirna is transfected, the amount should be increased accordingly. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. Rnai in cultured mammalian cells using synthetic sirnas. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died.
Lipofectamine 2000 is commonly used for sirna transfections. The product manual includes advice on how to optimize transfection efficiency, as a number of conditions can be varied including dna. For optimal transfection conditions with the mission sirna transfection reagent, the for 1. I would like to transfect 293t cells with lipofectamine 3000. A sample protocol is listed here for transfection experiments performed in 6well plates. Jul 24, 2007 advanced transfection with lipofectamine 2000 reagent. The reagent provides high transfection efficiency in many cells types and formats. Plate cells so they will be 7090% confluent at the time of transfection.
Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Lipofectamine 2000 transfection reagent from invitrogen. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells. Lipofectamine 2000 transfection reagent dartmouth college. Transfection guide overview of transfection methods promega.
However, few studies have examined cellular responses to this delivery system. Lipofectamine 2000 transfection protocol 10cm plates reagents. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. May 21, 2018 c representative confocal images of ls174t spheroids showing localization of controlcy3 sirna formed in optimem using lipofectamine 2000 at 2, 6 and 24 hours post transfection. By use of this product, you accept the terms and conditions outlined in the licensing and warranty statement contained in this user manual. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Tm328 fugene hd transfection reagent technical manual. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
It consistently produces high transfection efficiency and high protein overexpression. Rnai or technical service, as are celltype specific rnai transfection protocols see rnai protocols. 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. Lipofectamine transfection of c2c12 cells bridges lab. Lipofectamine 2000 dna transfection reagent protocol. The sirna transfection reagent is vided as a sterile solution, and is compatible with serum and antibiotics. I use 24well plates for transfections in triplicates. Mix gently and incubate for 20 min at room temperature to allow the sirna. Lipofectamine 2000 reagent protocol thermo fisher scientific. I want to try to use hela cell and lipofectamine 2000 to do. Mission sirna transfection reagent s1452 technical. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate.
Xtremegene sirna transfection reagent polymer reagent for. Product is guaranteed for 6 months from the date of shipment if stored properly. Effects of lipofectamine 2000sirna complexes on autophagy. Transfection protocol lipofectamine 3000 transfection reagent. Mission sirna transfection reagent s1452 technical bulletin. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added.
Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. As always, use good cell culture practices and wear your personal protective equipment. Prepare 4 tubes each with 50 microliter of optimem medium, and label them 1 to 4. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Transfection protocol lipofectamine 3000 transfection. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. 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. 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. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. I usually use 56 ulwell lipofectamine 2000 for an sirna concentration of 150 nm in a 24well plate as suggested by the vendor. Prewarm 50ml of optimum stored in cold room at 4c 10min. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25. Lipofectamine rnaimax reagent protocol thermo fisher scientific.
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. Xtremegene sirna transfection reagent polymer reagent. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Advanced transfection with lipofectamine 2000 reagent. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. Methods volume 33, issue 2, june 2004, pages 95103. Effects of lipofectamine 2000sirna complexes on autophagy in.