Transient plasmid dna transfection into hela and hacat. Maintain p19 cells undifferentiated in mem with 10% serum 7. Cellspecific transfection protocols thermo fisher scientific us. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Transfection of ht29 cells with sirna and lipofectamine 2000. Does lipofectamine 2000 give a stable transfection. 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. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. I am getting shitty transfection efficiency using effectene and geneporter 2 transfection reagent. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Mdm2 promotes epithelialmesenchymal transition and. Optimized conditions for successful transfection of human.
Ht 29, human colon adenocarcinoma, plasmid dna, lipofectamine 3000. The following cell lines have been successfully transfected with the indicated transfection reagents. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Xtremegene sirna transfection reagent polymer reagent. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Invitrogen lipofectamine 3000 transfection reagent 0.
Lipofectamine transfection of c2c12 cells bridges lab. Transfection efficiency comparison in hela cells using transfectamine. Xtremegene sirna transfection reagent efficiently delivers short interfering rna sirna into many commonly used cell types including hela, nih 3t3, hek293, chok1, and cos7, and several hardtotransfect cell lines, such as ht29, a human adenocarcinoma cell line. What would be the simplest possible protocol to lyse cells for genomic pcr, with.
Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338 500 pdf. Scaling up or down lipofectamine 3000 reagent transfections. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Plate cells so they will be 7090% confluent at the time of transfection. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. Superior performance for cotransfection of sirna and plasmid dna. Highest transfection efficiency in cells at 90% confluency.
Can anybody recommend a method transfection substrate of how to transfect ht29 cells. Livecell imaging to compare the transfection and gene. Help on transfecting mcf7 and hct 116 cells transfection. Role of specific endocytic pathways in electrotransfection of cells. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The microscopic images were manually analyzed to quantify. Transfecting a human neuroblastoma cell line with monster. I have successfully transfected hct116 cells, but have been told ht29s are more difficult to transfect. Altogen biosystems provides in vivo transfection reagents, over 100 preoptimized in vitro transfection kits for cell lines and primary cells, and electroporation delivery products. Highest transfection efficiency in many cell types and formats. Using lipofectamine 2000 cd reagent for transfection provides the following advantages. Sep192012 hi everyone, can anyone suggest a protocol for transfection of ht29 cells with sirna using lipofectamine 2000. Multipurpose reagent most suitable for pdna transfection, suitable for.
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. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. This protocol is for transfecting plasmid dna into mammalian cells. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Invitrogen lipofectamine 2000 transfection reagent. Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by natalie betz, ph.
Invitrogen lipofectamine 3000 transfection reagent. I use 24well plates for transfections in triplicates. Transfection protocol lipofectamine 3000 transfection. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension. Higher densities can lead to spontaneous differentiation. This reference provides a recommended procedure to transfect plasmid dna into ht 29, human colon adenocarcinoma. The reagent provides high transfection efficiency in many cells types and formats. Test serumfree media formulations for compatibility with lipofectamine. To transfect cells with sirna, follow the protocol as described for dna but.
With the experimental protocol described in the materials and methods. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng p3000 reagent per well 0. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. Scientific, and imaged with the is2000mm image system carestream. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. This reference provides a recommended procedure to transfect plasmid dna into ht29, human colon adenocarcinoma cells atcc no. Transfectamine 5000 transfection reagent is a powerful and versatile. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the transfection of dna into eukaryotic cells with low cytotoxicity. Lipofectamine 2000 cd chemically defined transfection reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Optimemin ul, equal volume for dna and lipofectamine lipofectaminein ul 25.
Highest transfection efficiency in many cell types and formats e. Altogen cro offers in vivo rnai services, tumor xenograft models, toxicology testing, stable cell line generation, and. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Invitrogen lipofectamine 3000 transfection reagent l3000008 gibco optimem i reduced serum medium 31985062 thermo scientific nunc 24well cellculture treated multidishes 142475 step tube complexation components amount per well 24well. Lipofectamine 2000 dna transfection reagent protocol. A highly efficient method of transfecting hep g2 cells.
Viafect reagent offers performance similar to or better than that of lipofectamine 2000 reagent, and it may be suitable for. Reverse transfection is the transfer of genetic material into cells and is reverse because the order of dna and cells is reverse that of conventional transfection. Lipofectamine 2000 transfection reagent from invitrogen. Transfection reagents are highly efficient for dna and sirna transfection in vivo and in vitro. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Follow manufacturers handbook protocol unless otherwise indicated. Lipofectamine transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. Transfection using effectene, lipofectamine, and polyfect reagents many reagents are now available for optimization of cell transfection efficiency. Transient plasmid dna transfection into hela and hacat cells.
It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Immediately after electrotransfection, hek293, ht29, and hct116 cells. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. 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.
Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Transfection of mcf7 with lipofectamine 2000 maartje vogel. Protocol for ht29 sirna transfection using lipofectamine 2000. Transfection guide overview of transfection methods promega. The following is a general protocol for use of dharmafect transfection. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Protocol for ht29 sirna transfection using lipofectamine. Transfection p19 cells with lipofectamine 2000 the same protocol can be used for 293t cells 1. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. 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.
This result shows that simple, lipidmediated transfection of hep g2 cells is a viable alternative to gene. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. The use of these different lipofectamine 2000 amounts for transfection showed no significant differences. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of. Invitrogen transfection reagent, recommended transfection sku, protocol. Transfection protocol transfection components component cat.
Achieve superior transfection efficiency while maintaining low toxicity lipofectamine 3000 reagent maintains a high transfection efficiency within a robust dynamic range of lipid doses for quick and easy optimization. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Lipofectamine 2000 reagent thermo fisher scientific. Genscript recommends using lipofectamine 2000 for all transfections. A sample protocol is listed here for transfection experiments performed in 6well plates. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Dharmafect volumes per well represent guidelines and need to be optimized. I am using lipofectamine 2000 with optimem to transfect hek cells, but i havent had much luck so far. Lipofectamine 2000 is a very efficient transfection agent, the stability of your knockdown or your overexpression is only related to the half life of your sirna, plamid in the cell. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. The pcmvmyc3mdm2 plasmid or small interfering rna was transfected into cells using oligofectamine invitrogen, 12252011 or lipofectamine 2000 invitrogen, 11668019 according to the.