The delivery of dna into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. The impact of transfection mediated toxicity mirus bio. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Optimisation of dna transfection of the c2c12 muscle myoblast cell line, comparing use of polyethylenimine pei and lipofectamine2000.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Genscript recommends using lipofectamine 2000 for all transfections. However, few studies have examined cellular responses to this delivery system. For more tips for your rnai experiment, refer to seven steps to rnai success.
A sample protocol is listed here for transfection experiments performed in 6well plates. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity. Do any one has protocol for stable transfection with lipofectamine 2000. Yous uggested to thaw a new vial of cells if the cells had reached confluency even if it was few passages before. How does transfection with lipofectamine 2000 work. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. But muscle, for example, will be hard to transfect. Lipofectamine transfection of c2c12 cells bridges lab. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. Mouse neuronal cells were transfected with a gfpexpressing plasmid using either lipofectmax transfection reagent left, or lipofectamine 2000 right. 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. See page 2 to view a typical dna transfection procedure.
Both lipofectamine 2000 and 3000 work well for 293t cells, you can just follow their protocol. Viafect transfection reagent is a highperforming, lowtoxicity reagent. Invitrogen lipofectamine 3000 transfection reagent fisher. Transient plasmid dna transfection into hela and hacat cells. May 11, 2016 it is widely accepted that the final transfection efficiency te of lipidbased transfection reagents trs is ratelimited by several biological barriers such as cellular uptake, intracellular trafficking, endosomal escape and nuclear entry 1,2,3,4,5,6,7,8.
Optimising c2c12 muscle myoblast transfection with. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Plate cells for 212hrs but not over 24 hours prior to transfection to achieve a 4080% confluence at the time of transfection. Systematic screening of commonly used commercial transfection. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount.
Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338 500 pdf. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. I use 24well plates for transfections in triplicates. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Viafect transfection reagent, lipofectamine 2000 transfection reagent or lipofectamine 3000 transfection reagent following manufacturer instructions, applying either 5l. 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. Invitrogen lipofectamine transfection reagent protocols have been. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Lipofectamine 3000 transfection reagent kit youtube.
Does anyone have experience with the lipofectamine 2000 toxicity. Lipofectamine 2000 is commonly used for sirna transfections. On the reliability and validity of manual muscle testing. Does it mean that once hek293 cells reached 100% confluency, it cannot be used at all for transfection expts. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf.
Cellspecific transfection protocols thermo fisher scientific us. Invitrogen lipofectamine 3000 transfection reagent. Lipofectamine 3000 has two main advantages over our existing reagent, the biggest one is its significantly higher transfection efficiency in difficult to transfect cell lines. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for huh7 and vero cells. Reverse transfection protocol for dna in 96well plates using transitx2 dynamic delivery system. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Start any new transfection by testing the recommended four. The intracellular trafficking mechanism of lipofectamine. Prewarm 50ml of optimum stored in cold room at 4c 10min. Invitrogen lipofectamine 2000 transfection reagent life. If you still have this tox effect, then you should think about testing polymerbased reagents. The reagent provides high transfection efficiency in many cells types and formats.
Add 6l of lipofectamine2000 from biostores and 100 l of. I follow the standard recommended testing procedures and protocol for transfection. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Invitrogen lipofectamine 2000 transfection reagent. Lipofectamine 2000 transfection protocol 10cm plates reagents. Troubleshooting for transfection of the crisprcas9 using. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Transfection efficiencies were assessed by gfp signal. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Effects of lipofectamine 2000sirna complexes on autophagy in.
The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Superior performance for co transfection of sirna and plasmid dna. What percentage confluency should be used for stable transfection. An example of high efficiency forward and reverse transfection of a549 cells using the broad spectrum transitx2 dynamic delivery system can be found in the figure below. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1 and with the. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Studies were undertaken to evaluate and compare the transfection efficacy. 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. Lipofectamine plasmid transfection bridges lab protocols. You can find in the instruction manuals of these two reagents a simple way of doing so. 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. One day before transfection, plate cells in growth medium without antibiotics so that they will be 9095% confluent at the time of transfection 0.
For additional information on scaling your transfection reaction, see page 4. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Specially designed cationic lipids, such as lipofectamine transfection reagents, facilitate. I have used both lipofectamine 2000 and 3000 and always get the same results. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Nov 20, 2008 i have a question regarding confluency of hek293 cells for transfection. Does anyone have experience with the lipofectamine 2000. Invitrogen lipofectamine 2000 transfection reagent fisher.
Lipofectamine 2000 reagent thermo fisher scientific. Transient transfection of mammalian cells with lipofectamine. The impact of transfection mediated toxicity gene expression and cytotoxicity analysis of transfection reagents introduction while plasmid dna delivery is a widely used method to study cellular functions of proteins of interest, studies to identify nontoxic gene delivery reagents are limited. 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. When a finite cell line undergoes transformation and acquires the ability to. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Lipofectamine 2000 transfection reagent from thermo fisher. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. 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. Clinical laboratory products microsatellite instability testing.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Comparison of lipofectmax and lipofectamine 2000 in plasmid transfection on mouse neuronal cells. General procedure for transfection of mammalian cells with. For example, liver and kidneyderived cell lines may be more suitable for toxicity testing. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic. Numerous chemical and physical approaches have been used to deliver the dna, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting. Different cell confluencies, dnareagent ratios and total transfection volumes were optimized for two chemical reagents including jetpei and lipofectamine 2000. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Lipofectamine 2000 dna transfection reagent protocol. In tube a place 2 g of dna in optimemi for a final volume of 200 l. I am about to start my stable transfections with lipofectamine 2000.
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