I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Different types of transfection reagents can be used for transfection, optimize the amounts of dna and liposomes for each type. 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. Samples were run on a 412% trisbis nupage gel invitrogen catalog no. Lipofectamine 2000 transfection reagent is a proprietary formulation. Based on the negative control, in which we applied puromycin to wildtype pk15 cells, we determined that the selection completed in 3 days. Lipofectamine reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Lipofectamine 2000 is a proprietary formulation for the transfection of. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency.
Highest transfection efficiency in many cell types and formats e. This product is covered by one or more limited use label licenses see the invitrogen catalog or our website. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen. 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. 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. Combine diluted dna and lipofectamine, mix and incubate at room temperature for 20 min. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine transfection of c2c12 cells bridges lab. However, rock inhibition has no effect on adherens junction formation in mdck or hct116 cells, suggesting that. Does anyone have experience with the lipofectamine 2000 toxicity.
Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. In a majority of cases, one wants the highest level of transfection possible with the lowest amount of cell death. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. 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. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells in a 24well format. What links here related changes upload file special pages permanent. Lipofectamintm 2000 invitrogen, dnaplasmid of interest, medium full medium, reduced medium, minimal medium, 24well plate procedure. Invitrogen lipofectamine 2000 transfection reagent. Use the recommended lipofectamine 2000 cd reagent amount. 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. Pool dishes of cells that have been prepared for transfection.
The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. For ae cultures and neuroblastoma cell lines, the same protocol was applied to cells. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may. Lipofectamine 2000 cd reagent thermo fisher scientific. For other formats, see scale up or down transfections. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies. The mtt cell proliferation assay was performed using kit from fluka, ltd. The rho target prk2 regulates apical junction formation in. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 lf2000 is a reagent that i have found fits exactly what i need. We have had success using lipofectamine reagents in the past and the lipofectamine 3000 appears to be the best option. Lipofectamine 2000 transfection reagent thermo fisher scientific.
The first one describes my very own handling of the protocol. 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. Life technologies corporation andor its affiliates warrant their products as set forth in the life. Detailed information is provided in additional file 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. We also report on celltype specific transfection efficiency of neurons and astrocytes. Optimizing transfection lipofectamine 2000 cd reagent. The trusted, simple, broadspectrum transfection reagent for most cell lines. Transfection commonly refers to the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells.
I use 24well plates for transfections in triplicates. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene. Invitrogen lipofectamine 3000 transfection reagent 1. Does anyone have experience with the lipofectamine 2000. 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. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
Mscs transfected by lipofectaminetm 2000 invitrogen, grand island, ny, usa were used as controls. Double transfection of keratinocytes with lipofectamine 2000 invitrogen equimpent. Systematic screening of commonly used commercial transfection. Original article an improved method for increasing the efficiency of. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 2000 dna transfection reagent protocol. The protocal is from thermo fisher scientific important guidelines for transfection 1. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3.
Stealthsirna transfection protocol lipofectamine 2000 thermo. Outline of protocol steps for the use of lipofectamine 2000 reagent. Lipofectaminez 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates. Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents.
However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance. Protocol for lipofectamine transfection in nih3t3 cells. I personally have used this reagent to transfect cells for fluorescent microscopy, gst pull downs, and coimmunoprecipitation experiments.
The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000 reagent thermo fisher scientific. For each transfection, add 1 l lipofectamine 2000 to 25 l optimem in a 1. It is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours. Warm rawgm1 10 ml to 37 c and equilibrate in 5% co2 incubator. Adjust the amounts of components according to your tissue culture format. Original article a comparative study of transfection of. Use the following procedure to transfect mammalian cells in a 24 well format.
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. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Supplementary material for aktmediated regulation of autophagy and tumorigenesis through. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Invitrogen lipofectamine 3000 transfection reagent. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. For additional information on scaling your transfection reaction, see page 4.
Two days after transfection, cells were lysed in nonidet p40 lysis buffer 1%nonidet p40, 20 mm trishcl, ph 7. Cells in a 6 well dish, plated and at 70% confluence. Day 2procedure for preparing lipofectamine 2000 and dna 1. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Lipofectamine 2000 transfection protocol 10cm plates reagents. I have used lf2000 successfully with the following cell lines. Lipofectamine reagents go online to view related products. 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 has been used successfully to transfect synthetic short. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Estimating transfection efficiency in differentiated and. Protocol for dna transfection and luciferase assay using.
It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. 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. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. 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. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Dnalipofectamine complexes must be made in serumfree medium such as optimem reduced. Advanced transfection with lipofectamine 2000 reagent. Lipofectamine 2000 from invitrogen biocompare product. Does anyone know the lipofectamine concentration for transfection. System biosciences using lipofectamine 2000 invitrogen. By the use of this product you accept the terms and conditions of all applicable limited use label licenses. Lipofectamine 2000 invitrogen, usa in optimem medium according to manufacturers instructions. I am performing transfection on mcf7 cells using lipofectamine 3000 invitrogen but unable to get any transfectants, instead.