Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. On the day of transfection, complexes are prepared in a multiwell plate and incubated for the recommended complex formation time. Why is only lipofectamine the preferred way to introduce sirnas. 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. Add 6l of lipofectamine2000 from biostores and 100 l of. Thee two most important factors in choosing transfecting. Advanced transfection with lipofectamine 2000 reagent. Rnai in cultured mammalian cells using synthetic sirnas.
Prepare plasmid dnalipid complexes recommend 2 doses of lipid. I usually use 56 ulwell lipofectamine 2000 for an sirna concentration of 150 nm in a 24well plate as suggested by the vendor. Jan 18, 2011 primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as nonalcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. Freshly passaged cells are then directly added to the multiwell plates containing the transfection complexes at twice the density of a standard forward trans. Mix gently and incubate for 20 min at room temperature to allow the sirna. We have very often used hiperfect and microporation with good success rate for sirna transfection even though for the same cell lines, dna transfection is better achieved with lipofectamine. Cotransfection of two plasmids need the protocol for lipofectamine 2000 based transfection nov 012009 hi, i am trying to cotransfect transient trasnfection two different plasmids in to hek293t cells. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. I normally incubate my cells in media with antibiotics and serum, and a few hours before transfection i aspirate the media and replace with antibiotic free dmem with serum.
Thee two most important factors in choosing transfecting reagent are. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 2000 and lipofectamine rnaimax transfection. After a 5 minute incubation in optimem, the lipofectamine 2000 and dna are combined and incubated for 20 minutes with gentle shaking at room temperature. This active endosome escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. I use 24well plates for transfections in triplicates.
To obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. I am using lipofectamine 2000 with optimem to transfect hek cells, but i havent had much luck so far. Stealthsirna transfection protocol lipofectamine 2000. However, few studies have examined cellular responses to this delivery system. Product is guaranteed for 6 months from the date of shipment if stored properly. For example, for cotransfection of plasmid dna and sirna in a 24well plate, we would recommend using. Resuspend the sirna in h 2 o to a concentration of 100. Invitrogen lipofectamine 3000 transfection reagent 1. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. 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. Effects of lipofectamine 2000sirna complexes on autophagy in. A hela cells were transfected with hcv dsrna using lipofectamine 2000 reagent.
Lipofectamine 2000 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Cotransfection of two plasmids tissue and cell culture. Dsirnas were originally developed as a collaborative effort with dr john rossi of the beckman research institute of the city of hope. Effects of lipofectamine 2000 sirna complexes on autophagy in hepatoma cells. Plasmid dna, synthetic sirna, rnai plasmids shrna, mir content and storage contains one vial of lipofectamine 3000 reagent 1. Lipofectamine 2000 transfection reagent from invitrogen. The indicated sirna plasmids and the antisense oligodeoxynucleotides were transiently transfected into vero e6 cells in 24well plates via lipofectamine 2000. Invitrogen lipofectamine 3000 transfection reagent 0. Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. The protocal is from thermo fisher scientific important guidelines for transfection 1. There is a need to identify simple methods to genetically manipulate primary hepatocytes and. The microscopic analysis was performed using antirigi. Nter sirna nanoparticlemediated knockdown of gene expression xtremegene 360 transfection reagent if additional help is needed, please email our technical services group.
For the cell lines easy to transfection, you can use either of the procedures reverse or forward. 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. To transfect cells with sirna, follow the protocol as. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. The vector contained an ofp reporter gene and was transfected. Lipofectamine 2000 reagent thermo fisher scientific. Troubleshooting for transfection of the crisprcas9 using. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Why is only lipofectamine the preferred way to introduce. Nucleic acidlipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum.
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. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Fluorescently labeled sirna allows simply to monitor sirna delivery. Effects of lipofectamine 2000sirna complexes on autophagy. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Invitrogen lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Add the oligomer lipofectamine 2000 complexes from step 2. Has anyone tried sirna transfection with lipofectamine3000. Lipofectamine 2000 procedure for simultaneously transfecting. Lipofectamine 2000 transfection reagent dartmouth college. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Kd effect of highly efficient target gene is verified.
To determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. View and download toro lawn boy service manual online. Optimized conditions for successful transfection of human. Broad spectrum dna sirna delivery one transfection reagent and protocol for a variety of cells. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. The sirna usually lasts for about 5 days so it is not permanent, same as lipid carriers. Lipofectamine 2000 is commonly used for sirna transfections. Jun 10, 2011 lipofectamine 2000 is commonly used for sirna transfections. Manual reel lawn mowers lawn mowers the home depot. After the 5min incubation, combine the diluted sirna with the diluted lipofectamine 2000 total volume is 100. Fullfeature push reel mower 41516s, 5blade roller assembly mowers cutting height adjusts from 12 in. 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.
Lin28 is an rnabinding protein that is highly expressed in undifferentiated hescs and is among four factors including oct4, sox2, and nanog used for making ipscs yu et al. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Prewarm 50ml of optimum stored in cold room at 4c 10min. I have used both lipofectamine 2000 and 3000 and always get the same results.
Highest transfection efficiency in many cell types and formats e. In addition, analysis is performed to ensure that the chosen sites do not target alternatively spliced exons and do not include. I would like to transfect 293t cells with lipofectamine 3000. Invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. 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. First, we used a sirna specific for lin28 silin28 qiu et al. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Lipofectamine 2000 transfection protocol 10cm plates reagents. Shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine 3000 reagent sirna lipofectamine 3000 reagent 96well 0. Manual walk behind reel lawn mower with attachable bagger and 9 position cutting heights. Photograph of expected results dna lipofectamine scaling up or down. Highly efficient sirna delivery into primary cultures of rat. Although this amount is good for a wide range of sirna concentrations, if more sirna is transfected, the amount should be increased accordingly.
Dilute sirnachimera with optimem i reduced serum medium invitrogen up to 50 l and vortex for a few seconds dilute appropriate amount of lipofectamine 2000 by optimem i reduced serum medium to 50 l and vortex for a few seconds for siha and skov3 cells, dilute 1. The sirna sequence has low homology to target genes. Comparative nucleic acid transfection efficacy in primary. Sirna and plasmid cotransfection using lipofectamine 3000. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Plate cells so they will be 7090% confluent at the time of transfection. Dsirnas are chemically synthesized, 27 nt rna duplexes that are optimized for dicer processing and are ideal for smallscale in vitro applications. Scaling up or down lipofectamine 3000 reagent transfections. These 27mer duplexes have increased potency in rnai compared to traditional 21mer sirnas. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Even so, mnhap was less toxic than lipofectamine 2000 as demonstrated in fig.
This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. Lipofectamine 2000 thermo fisher, xtremegene roche, hiperfect qiagen, and. Inspired by the above observations, we next set out to test sirnas in h1 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. As sirna itself had no obvious cytotoxic effect on bel7402 cells, the cytotoxicity of mnhap sirna and lipofectamine 2000 sirna complexes were quite possibly due to mnhap and lipofectamine 2000 themselves. It is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature. Virus delivery of shrna is permanent and you may be able to make stable cell lines. For each transfection sample, prepare oligomerlipofectamine 2000 complexes as follows. How does transfection with lipofectamine 2000 work. L cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0.
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