Use the recommended lipofectamine 2000 cd reagent amount. Dnalipofectamine complexes must be made in serumfree medium such as optimem reduced. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. However, rock inhibition has no effect on adherens junction formation in mdck or hct116 cells, suggesting that. The first one describes my very own handling of the protocol.
Advanced transfection with lipofectamine 2000 reagent. Lipofectamine reagents go online to view related products. For other formats, see scale up or down transfections. Invitrogen lipofectamine 2000 transfection reagent. Stealthsirna transfection protocol lipofectamine 2000 thermo. Combine diluted dna and lipofectamine, mix and incubate at room temperature for 20 min. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen. Detailed information is provided in additional file 1. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. 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. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency.
This product is covered by one or more limited use label licenses see the invitrogen catalog or our website. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies. Lipofectamine 2000 has been used successfully to transfect synthetic short. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Samples were run on a 412% trisbis nupage gel invitrogen catalog no. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. It is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours. Lipofectamine 2000 invitrogen optimem media gibco 6well polylysine coated plates mini production 100 mm plates polylysine coated plates for small production 150 mm plates polylysine coated plates for large production retroviral vector plasmid media. Pool dishes of cells that have been prepared for transfection. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. System biosciences using lipofectamine 2000 invitrogen.
Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest variety of adherent and suspension cell lines. Lipofectamine reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Supplementary material for aktmediated regulation of autophagy and tumorigenesis through. Mscs transfected by lipofectaminetm 2000 invitrogen, grand island, ny, usa were used as controls. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Optimizing transfection lipofectamine 2000 cd reagent. The trusted, simple, broadspectrum transfection reagent for most cell lines. The intracellular trafficking mechanism of lipofectamine. Warm rawgm1 10 ml to 37 c and equilibrate in 5% co2 incubator.
Outline of protocol steps for the use of lipofectamine 2000 reagent. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Invitrogen lipofectamine 3000 transfection reagent. For each transfection, add 1 l lipofectamine 2000 to 25 l optimem in a 1. Invitrogen lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect a variety of payloads into the widest.
Double transfection of keratinocytes with lipofectamine 2000 invitrogen equimpent. Lipofectamine 2000 dna transfection reagent protocol. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Lipofectamine 2000 is a proprietary formulation for the transfection of. 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.
Lipofectamine 2000 reagent thermo fisher scientific. The protocal is from thermo fisher scientific important guidelines for transfection 1. Lipofectamine 2000 transfection protocol 10cm plates reagents. Protocol for lipofectamine transfection in nih3t3 cells. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Adjust the amounts of components according to your tissue culture format. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells in a 24well format. Day 2procedure for preparing lipofectamine 2000 and dna 1. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. I have used lf2000 successfully with the following cell lines.
We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. Original article a comparative study of transfection of. 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. Invitrogen lipofectamine 3000 transfection reagent 1. Two days after transfection, cells were lysed in nonidet p40 lysis buffer 1%nonidet p40, 20 mm trishcl, ph 7. Does anyone have experience with the lipofectamine 2000 toxicity. I am performing transfection on mcf7 cells using lipofectamine 3000 invitrogen but unable to get any transfectants, instead.
Cells in a 6 well dish, plated and at 70% confluence. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine 2000 transfection reagent is a proprietary formulation. Lipofectaminez 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates. Lipofectamine 2000 cd reagent thermo fisher scientific. For ae cultures and neuroblastoma cell lines, the same protocol was applied to cells. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may. Original article an improved method for increasing the efficiency of.
Estimating transfection efficiency in differentiated and. Different types of transfection reagents can be used for transfection, optimize the amounts of dna and liposomes for each type. What links here related changes upload file special pages permanent. Does anyone know the lipofectamine concentration for transfection. 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. We also report on celltype specific transfection efficiency of neurons and astrocytes. Protocol for dna transfection and luciferase assay using. Life technologies corporation andor its affiliates warrant their products as set forth in the life. Does anyone have experience with the lipofectamine 2000. Highest transfection efficiency in many cell types and formats e.
The rho target prk2 regulates apical junction formation in. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene. Lipofectamine 2000 lf2000 is a reagent that i have found fits exactly what i need. Invitrogens lipofectamine 2000 is highly cited in the literature, a sign of a successful reagent, so when our lab embarked upon the path to develop a successful transfection protocol, we included lipofectamine 2000 along with the other leading reagents. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Transient transfection assays were conducted using the lipofectamine 2000 transfection reagent invitrogen following the manufacturers instructions, with four replicate wells per condition per experiment in at least three in. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone has found an optimal lipo 3000 dna ratio for this cell line. Systematic screening of commonly used commercial transfection. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to deliver invitrogen geneart precision tals and crispr nucleases targeting the aavs1 safe harbor locus in u2os and hepg2 cell lines in 12well format.
Lipofectamintm 2000 invitrogen, dnaplasmid of interest, medium full medium, reduced medium, minimal medium, 24well plate procedure. By the use of this product you accept the terms and conditions of all applicable limited use label licenses. In a majority of cases, one wants the highest level of transfection possible with the lowest amount of cell death. The mtt cell proliferation assay was performed using kit from fluka, ltd. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2.
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