Tips and tricks on how to improve transfection experiments. Comparison of transfection efficiency of nonviral gene. General considerations successful transfection involves optimizing the fugene hd transfection reagent. In 6cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. Fugene hd transfection reagent technical manual promega. Xtremegene hp dna transfection reagent is a high performance reagent that transfects a broad range of eukaryotic cells, including insect cells and hardtotransfect. It is suitable for use in media with or without serum, and for transient or stable transfection, as well as cotransfections of multiple dna plasmids. Fugene hd transfection reagent for efficient mammalian. Three microliters of fugene 6 transfection reagent is combined with 12 g of reportergene vector dna, and used to transfect cos1 cells.
May 22, 20 this feature is not available right now. Symbols are used in this instruction manual to highlight important. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetpei and expressfect. Xtremegene transfection reagents from roche have been used for a wide range of applications and a vast array of cell lines, primary cells and stem cells. Fugene hd transfection reagent is a proprietary, nonliposomal, low toxicity formulation designed for high efficiency delivery of dna into a broad range of cell lines. For further improvement of transfection efficiency refer to transfection optimization section of this manual. Vortex and incubate the mixture at room temperature for 15 min before. In animal cells, transfection is the preferred term as. Comparison of commercial transfection reagents optimized for delivery of episomal vectors into primary cells we compared the transfection efficiency and cytotoxicity of the transfection reagentepisomal vector complexes derived from the lipofectamine 2000, fugene hd and jetpei at their optimum ratios le 4, fe 4 and je 2. Enhanced performance in a range of cell types, including difficulttotransfect cell lines.
The fugene hd transfection reagent is a nonliposomal formulation designed to transfect dna into a wide variety of cell lines with high efficiency and low toxicity. In the above experiment, 48hour survival was measured on the cells with or without fugene hd transfection. First, not every transfection reagent is perfect for every cell line. Fugener hd transfection reagent technical manual tm328. Xtremegene hp dna transfection reagent is a multicomponent reagent that forms a complex with dna, then transports the complex into animal or insect cells. Fugene hd transfection reagent manual active motif. Fugene 6 transfection reagent is gentle on the cells. Combinational use of lipidbased reagents for efficient transfection. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.
While considering a suitable transfection reagent, it is. Apr 25, 2018 thus, among the reagents tested, fugene hd transfection reagent impacted the fewest number of transcripts implicated in the regulation of the antigen processing pathway n 5, all other transfection reagents resulted in the differential expression of eight transcripts. Maximal transfection efficiency in mc3t3e1, c3h10t12, hela, c2c12, hep g2, and hct116 was seen using fugene hd, in pt30, 4t1, and hekn was seen using arrestin, and in mcf7 was seen using jetpei. Use 2g dna and 4l fugene per 10cm dish scale this up to prepare in bulk. Fugene hd transfection reagent promega corporation. Fugene hd transfection reagenta is a novel, nonliposomal formulation designed to transfect dna into a wide variety of cell lines with high efficiency and low. A preparation of cells for transfection use cells that are healthy and proliferating well. This study compared six commercially available reagents arrestin, expressfect, fugene hd, jetpei, lipofectamine 2000, and superfect for gene transfection. Fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. Fugene hd transfection reagent offers unrivaled transfection performance with minimal impact on cell physiology jacobson et al. Vwr offers transfection reagents for numerous applications, including bioproduction, in vivo work, viral production, dna and sirna transfection, crisprcas 9 and more. The database includes over 25 cancer models and provides transfection protocols specific for each cell line. Promega offers three premium transfection reagents. Dna complex for a minimum of 20 min at room temperature no longer than 45 min though.
Avoid contact of undiluted fugene hd transfection reagent with the sides of the tube or plate. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Biorad, hercules, ca, and fibro blast transfection reagent altogen. Fugene hd transfection reagent lets you work in your cancer model. We routinely use fugene hd to introduce plasmids encoding tagged lipidbinding domains into raw264. Fugene 6 transfection reagent is a nonliposomal formulation designed to transfect plasmid dna into a wide variety of cell lines with high efficiency and low toxicity. Xtremegene hp dna transfection reagent highperformance. Use the table below to find the right product for your experiment. This reagent combines high transfection efficiency with exceptionally low cytotoxicity.
This reagent is suitable for use in media with or without serum. Fugene 6 reagent complex prior to plating, making it a strong candidate for high throughput applications. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. Fugene transfection promega reagents fugene promega e2311 is a nonliposomal transfection reagent designed to work, with high efficiency and low toxicity, in a wide variety of cell lines. The protocol does not require removal of serum or culture medium and does not require washing or changing of medium after introducing the reagentdna complex. For each cell line, the maximum rlus were set at 100% for one reagent and the other 3 were judged relative to that reading. Fugene hd transfection reagent quick protocol fb112pdf 99 kb english. Fugene hd transfection reagent is a nonliposomal formulation designed to transfect dna into a wide variety of cell lines with high efficiency and low toxicity. Fugene hd transfection reagent quick protocol fb112pdf.
Transfection of sf9 cells in suspension nih common fund. Always mix fugene 6 reagent prior to use vortex for one second or use inversion. Bring fugene hd transfection reagent to room temperature, mix well. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than invitrogen lipofectamine 2000 transfection reagent and fugene hd reagent for all five cell lines. Combine the reagents in table 2 for each transfection. Fugene hd transfection reagent for efficient mammalian cell. Additionally, low cell numbers can be transfected in 96well plates. Conduct at least three replicate transfections per construct in each condition e. Fugene hd transfection reagent is a proprietary blend of lipids and other components. The use of fugene hd does not require culture medium change or serum removal during. We examined the efficiency and cytotoxicity using nine different cell lines mc3t3e1 mouse preosteoblasts, pt30 human epithelial precancer cells, c3h10t12 mouse stem cells, mcf7 human breast cancer cells, hela human cervical.
Avoid contact of undiluted fugenehd transfection reagent with the sides of the tube or plate. With fugene hd, there is no need to change media after an. Dna ratio, which is optimal for most breast cancer cell lines. Before the experiment, i place the optimem at room temperature for 10min and vortex the fugene 6 to make it evenly. The best condition highest rlus was chosen for each transfection reagent. Add 3 ul fugene hd directly to the transfection medium, avoid touching the side of the tube when adding fugene. Dispase performed worse than tryple and edta, comparing to tryple. L overview fugene hd transfection reagent is a multicomponent reagent that forms a complex with dna, then transports the complex into animal or insect cells.
Fugene hd transfection reagent active motif lightswitch store. Optimized for use with all lightswitch plasmid products. Lipofectamine stem transfection reagent thermo fisher. All three offer broad spectrum transfection capability and low toxicity but the same question always comes up. Fugene hd transfection reagent can be used in transfection protocols in the presence of serum up to 100%, allowing transfection of cell types that require continuous exposure to serum, such as primary cell cultures. The protocol does not require removal of serum or culture medium and does not require washing or changing of medium after introducing the reagent dna complex. Cotransfecting plasmids how much transfection reagent. Before use, allow the vial of fugene hd transfection reagent to reach room temperature. Delivery of episomal vectors into primary cells by means. Lipofectamine 3000 reagent thermo fisher scientific sg. Achieve best results with reagents low cytotoxicity.
It is ideal for use in a variety of challenging applications, including transfection of cancer model cell lines, insect cells, stem cells, and for virus and protein production. Cells were also assayed for cell viability via and judged versus a no transfection control for each cell line. Perform transfection into hek293t as above using fugene hd roche applied bioscience and harvest these cells 48 h later for use in. Use the fugene hd protocol database to see if transfection conditions for your cell line are available. Designed for dramatically improved transfection efficiency, invitrogen lipofectamine stem transfection reagent offers a simple, robust and reproducible method for delivering dna, rna and rnp such as cas9grna complexes into a wide range of stem cells, including pluripotent stem cells, neural stem cells and mesenchymal stem cells figure 2. Fugene transfection protocol 1 add 3ul of fugene to 97ul of optimem, being sure to avoid touching the pipette tip to the side of the tube as the fugene will stick to plastic. According to manufactures protocol we can use 6ug of plasmid for 1. Adherent cells can be trypsinized and transfected by the dna. From this observation, we would predict that fugene hd transfection. Promega fugene hd transfection reagent fugene hd is a novel, nonliposomal reagent for transfecting dna into a variety of cell lines with high efficiency and low toxicity. Fugene hd transfection reagent is a multicomponent reagent that forms a complex with dna, then transports the complex into animal or insect cells. Fugene hd transfection reagent is a nonliposomal formulation designed.
The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. Dna ratio, amount of dna used, complexing time, cells and medium used, etc. Fugene 6 transfection reagent is stabilized for extended storage. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Scienceinsider,the new policy blog from thejournalscience, is your source for breakingnewsandinstant analysis from the nexus of politicsand science. Cotransfecting plasmids how much transfection reagent to use. It may also refer to other methods and cell types, although other terms are often preferred.
Fugene hd transfection reagent also can be used prediluted in transfection medium but should be used immediately. I want to do co transfection of 3 plasmids of size 8. Viafect transfection reagent transfection assay e4981. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in 96well format, and gfp expression was analyzed 48 hours posttransfection. Storage and stability fugene 6 reagent is shipped at room temperature.
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