Transfection of sf9 cells in suspension nih common fund. Use the table below to find the right product for your experiment. In 6cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. Transfection of j774 macrophages using fugene hd from roche based on preliminary experiments conducted by barish ozdamar in fred maxfields lab, fugene hd from roche consistently gives 510%. A protocol for nonliposomal transfection of dna into a variety of cell lines with high efficiency and low toxicity. Transfection protocol fugene hd transfection reagent instructions for use of products e2311 and e2312. Fugene hd transfection reagent quick protocol fb112pdf. The lipofectamine ltx and fugene hd lipidbased transfection reagents were. Fugene hd transfection reagent protocol promega corporation. It may also refer to other methods and cell types, although other terms are often preferred. Briefly vortex the xtremegene hp dna transfection reagent vial. Use the fugene hd protocol database to see if transfection conditions for your cell line are available. Dilute dna with appropriate diluent, such as serumfree medium to a final concentration of 1.
Fugene hd transfection reagent is a novel, nonliposomal formulation designed to transfect dna into a wide variety of cell lines with high efficiency and low toxicity. Few researchers will use the same cell type and same construct to generate data. Fugene 6 transfection reagent is gentle on the cells. Transfect cells according to the manufacturers protocol. Cultures were observed over 48 hours from start of transfection. Jul 15, 2016 the fugene hd transfection reagent protocol database has protocols for transfecting your cell line. Protocol 2 producing lentivirus in hek293t cells using a. Additionally, the fugene hd transfection reagent has been shown to support transfection in chemically defined. Search our fugene hd protocol database if youre new to the reagent or exploring the use of a new cell line. To a sterile tube or u or vbottom plate, add 90 98l of medium prewarmed to room temperature so that the final volume after.
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 multicomponent reagent that forms a complex. Protocol 2 producing lentivirus in hek293t cells using a 2. Gently drip 5l or 3l 96 or 384well formats onto seeded cells. Fugene hd transfection reagent for efficient mammalian. Fugene hd transfection reagent for efficient mammalian cell. Improving the success of your transfection promega connections. The fugene hd to plasmid dna ratio shown in this example is 3. The amount of dna and the fugene hd transfection reagent in protocol described bellow calculated to be sufficient to transfect cells in 20 wells of 96well. Fugene hd is added, the transfection mix is vortexed for 1 s, and then incubated for 15 min. 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 toxicity. The day before transfection plate adherent cells in the amount that by the time of transfection. The fugene hd protocol database is a collection of protocols that guide the transfection process for a variety of cell lines.
Promega corporation 2800 woods hollow road madison, wi 537115399 usa toll free. Fugene hd transfection reagent is a multicomponent reagent that forms a complex with dna, then transports the complex into animal or insect cells. This is the preferred protocol for use in highthroughput screening applications. A protocol for nonliposomal transfection of dna into a variety of cell lines with high efficiency and low. Fugene hd transfection reagent promega corporation. 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. Optimize transfection of cultured cells promega corporation. These protocols guide the transfection process for many cell lines, and will also calculate the volumes needed based on well format and the number of samples. Not every lab has a tried and true transfection protocol that can be used by all lab members. Fugene hd transfection reagent offers unrivaled transfection performance with minimal impact on cell physiology jacobson et al.
Before attempting this protocol, please make sure that. 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. Detecting ubiquitination of target proteins in live cells. Utilizes a protocol that does not require removal of serum or culture medium and does not require washing or changing of medium after introducing the reagentdna. Fugene is a transfection reagent that apparently increases transfection rates for animal cells. Add transfection mixture to wells with attached cells and express proteins overnight 1824 hours at 37c, 5% co 2. Fugene 6 transfection reagent is stabilized for extended storage. The protocol does not require removal of serum or culture medium and does not. Add transfection mixture to wells with attached cells and. Perform transfection into hek293t as above using fugene hd roche applied bioscience and harvest these cells 48 h later for use in. Storage and stability fugene 6 reagent is shipped at room temperature. Fugene is apparently a nonliposomal formulation of blended lipids, but its actual contents are not revealed anywhere.
Fugene hd transfection reagent is a nonliposomal formulation designed. Description the nanobret cmycmax interaction assayah monitors the interaction between human cmyc and max in living cells. We use it in lab for cell culture transfections, but i have no idea what it is or how it works. 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. You use the optimized reagents from the lightswitch luciferase assay system including the lightswitch luciferase assay reagents and fugene hd for best results. This reagent is suitable for use in media with or without serum, and for transient or stable transfection. For specific goclone transfection protocol examples, see our online.
Improving the success of your transfection promega. While considering a suitable transfection reagent, it is. Select the cell line, type of plate used and number of wells to be transfected and a protocol will be. Fugene 6 reagent complex prior to plating, making it a strong candidate for high throughput applications.
Promega offers three premium transfection reagents. Fugene hd transfection reagent lets you work in your cancer model. Fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. The use of fugene hd does not require culture medium change or serum removal during transfection experiment. 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.
Optimal transfection conditions for hek293 with the fugene hd reagent were determined using the protocol recommended in figure 2. All three offer broad spectrum transfection capability and low. Fugene hd transfection reagent quick protocol fb112pdf 99 kb english. Transitlt1 transfection reagent research reagent products. Description fugene 6 transfection reagenta is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high e. 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. The protocol can be scaled to produce different amounts of virus as needed. To a sterile tube or u or vbottom plate, add 9098l of medium prewarmed to room temperature so that the final volume after. This reagent is suitable for use in media with or without serum. Tips and tricks on how to improve transfection experiments. Detecting recruitment of target proteins to the proteasome.
Vwr offers transfection reagents for numerous applications, including bioproduction, in vivo work, viral production, dna and sirna transfection, crisprcas 9 and more. Add transfection mixture to wells with attached cells and express proteins for approximately 20 hours at 37c, 5% co 2. Additionally, low cell numbers can be transfected in 96well plates. Add 6l of fugene hd transfection reagent and incubate at room temperature for 10 minutes. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. The reagent is stable thro ugh the expiration date printed on the label when stored under these conditions.
Use 2g dna and 4l fugene per 10cm dish scale this up to prepare in bulk. These protocols were developed by promega corporation or fugent, l. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Transfection mixes are added dropwise to each well of the 6well plate, and the cells are incubated for 48 h in a humidified atmosphere at 37 c5% co 2.
Detecting the interaction of target proteins with e3. Has an easily optimizable protocol that allows it to transfect a wide range of eukaryotic cells, including animal and insect cells, with minimal cytotoxicity. Aug 30, 2019 not every lab has a tried and true transfection protocol that can be used by all lab members. Select the cell line, type of plate used and number of wells to be transfected and a protocol will be generated based on conditions tested by promega scientists. Cells were visualized 48 hours posttransfection and imaged. 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. Description 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. Add 3 ul fugene hd directly to the transfection medium, avoid touching the side of the tube when adding fugene. 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. 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. Fugene hd transfection reagent manual active motif. Always mix fugene 6 reagent prior to use vortex for one second or use inversion. 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.
The protocol does not require removal of serum or culture. Replating transfected hek293 cells into multiwell plates and adding halotag ligand 1. In animal cells, transfection is the preferred term as. Follow the transfection reagent protocol for seeding cells. 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. Fugene is a registered trademark of fugent, llc in the united states and other countries. Fugene6, and fugenehd are trademarks of fugent, llc.
Find the right product for your cells by using our selection guide. Lipofectamine stem transfection reagent thermo fisher. Detecting recruitment of target proteins to the proteasome in. Xtremegene hp dna transfection reagent sigmaaldrich. The use of fugene hd does not require culture medium change or serum removal during.