The fugene hd transfection reagent protocol database has protocols for transfecting your cell line. Adherent cells can be trypsinized and transfected by the dna. Fugene is apparently a nonliposomal formulation of blended lipids, but its actual contents are not revealed anywhere. Avoid contact of undiluted fugene hd transfection reagent with the sides of the tube or plate. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. The allinone system for the inducible expression of shrna. Fugener hd transfection reagent technical manual tm328. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. It may also refer to other methods and cell types, although other terms are often preferred. Apr 20, 2006 fugene 6 gives high efficiency transfection, yet relatively low altered endogenous cellular gene expression 20. In 6cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. All in vivo work with gl261 glioma cells was done in c57bl6 wildtype mice charles river laboratories, wilmington, ma, usa. Find msds or sds, a coa, data sheets and more information. 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.
Transfection reagent selection guide choosing a transfection reagent is easier when you can rely on the work of researchers who have used it before you. A protocol for the fugene 6 transfection reagent, a nonliposomal reagent that transfects dna into a wide variety of cell. Production of retroviruses using fugene6 32100 1 on the day before transfection, plate 0. Fugene 6 transfection reagent technical manual promega. Additionally, low cell numbers can be transfected in 96well plates. Product manual aav2 helper free packaging system catalog number. Lipofectamine2000 works quite well in 293cells 90% confluent in my. Many signal transduction events and transcription factors have been demonstrated to be associated with er stress. Karcher br 5560 w ep instructions manual pdf download. Citation list of established cells transfected with fugene 6. Prepare transfection mixture of plasmid dna and fugene transfection reagent roche according to manufacturers instructions. Always mix fugene 6 reagent prior to use vortex for one second or use inversion.
Guidelines for transfecting luciferase reporter vector with fugene 6. Fugene 6 transfection reagent a is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high e. Production of retroviruses using fugene 6 32100 1 on the day before transfection, plate 0. Gene promoter reporter vector user manual page 5 materials materials provided sequence information is available on our website. Lipofectamine 2000 was the most toxic reagent, with the highest cell death in the hek293 and hep g2 cell lines 55% loss in viability. Fugene 6 transfection reagent is gentle on the cells. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. Dna ratio, add 6l of fugene 6 transfection reagent to the medium, and mix immediately add fugene 6 transfection reagent directly to medium. Fugene 6 reagent complex prior to plating, making it a strong candidate for high throughput applications.
Profiling the expression pattern of gpi transamidase. While considering a suitable transfection reagent, it is. The cells are ready for transfection after 1820 hours, or when they are about 2530% confluent. In transfections, skbr3 cells were transfected with pmeebhapigs and pmeebgstgpi8 plasmids in the presence of the fugene 6 transfection regent. Study gene expression using the dualluficerase reporter dlrassay on the spectramax i3x multimode microplate reader with spectramax injector cartridge. One 1ml vial of fugene hd transfection reagent contains sufficient material to transfect over 3,000 wells in 96well cell culture plates at a 3. Lipofectamine 2000 transfection reagent was the most toxic, and produced as high as a 55% drop in viability in hek293 cells figure 2.
Hi,guys, currently im doing sirna transfection to hela cells with fugene 6. Citation list of established cells transfected with fugene 6 transfection reagent 10t12 timothy a. Promega fugene 6 transfection reagent 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. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. E to create a plate with titrations and replicates of different transfection conditions. Improving the success of your transfection promega connections. Plate slightly more cells when making vsvg pseudotyped viruses. Fugene 6 transfection reagent has been commercially available since 1997. 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. An example showing optimization of transfection conditions for the fugene hd reagent in a 96well plate is shown in figure 2. Gently shake the culture plate several times and harvest all media, including cells, in a sterile tube. Do not allow undiluted fugene 6 transfection reagent to contact the sides of the tube or u or vbottom plate.
Then the 293 cells were infected with ad5 moi 10 for helper function the cells. The use of fugene hd does not require culture medium change or serum removal during. Citation list of established cells transfected with fugene. Transfection the procedure is modified for using fugene 6 roche in a 12well culture plate. Fugene 6 transfection reagent has to be prepared in combination with serumfree dme medium. Improving the success of your transfection promega.
In animal cells, transfection is the preferred term as. Fugene 6 transfection reagent is a nonliposomal formulation designed to. Before the experiment, i place the optimem at room temperature for 10min and vortex the fugene 6 to make it evenly. To broaden the relevance of our findings, we employed another murine model where the tumor is initiated by the overexpression of pdgfb in nestinexpressing cells in vivo. In 6 cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. Olson 2000 activation of the myocyte enhancer factor2 transcription factor by calciumcalmodulindependent protein kinasestimulated binding of 1433 to histone deacetylase 5. Promega fugene 6 transfection reagent fugene 6 transfection reagent is a nonliposomal formulation designed to transfect plasmid dna into a wide. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Cell biolabs aav helperfree system allows the production of infectious recombinant human adeno.
This manual introduces a set of novel baculovirus transfer vectors that specifically enable multigene applications trowitzsch et al. Thank you so muchmcri tried both of them and lipofectamine 2000 was much betterin 293ft cells. Altered activity patterns of transcription factors induced. Additional items required items required but not provided transfection the procedure is modified for using fugene 6 roche in a 12well culture plate. Add 36 ul of fugene directly into 600 l of optimem. Vwr offers transfection reagents for numerous applications, including bioproduction, in vivo work, viral production, dna and sirna transfection, crisprcas 9 and more. The fugene hd transfection reagent is lipidbased, simpletouse and can yield high transfection efficiencies with minimal cytotoxicity.
Reagent wst1 roche applied science according to the instructions. Obtain optimal results with highsensitivity quantiation of luciferase activity with 6decade linear range. 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. However, after overnight incubation, almost 90% of cells are floating in the medium which are supposed to be dead cells. These transfer vectors are accompanied by modified recipient baculovirus dna that has been engineered towards improved protein production. This reagent is suitable for use in media with or without serum, and for transient or stable transfection. The concentration of sirna im using for transfection is 35nm which is within the range on the manual provided and much lower than some papers100nm.
The process in which er stress affects or interacts with other pathways is. The following figure demonstrates typical results seen with cell biolabs aav helperfree system. Fugene hd transfection reagent manual active motif. Stable cell line generation with pcoron1002egfpc1psld 32 7. Fugene 6 transfection reagent technical manualpdf 811 kb english.
Fugene 6, one of fugents premier products, is a multicomponent lipidbased transfection reagent. Fugene 6 transfection reagent is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high efficiency and low toxicity. All additional handling steps after the addition of the fugene 6 reagent. Storage and stability fugene 6 reagent is shipped at room temperature. The endoplasmicreticulum er responds to the burden of unfolded proteins in its lumen by activating intracellular signal transduction pathways, also known as the unfolded protein response upr. With fugene hd, there is no need to change media after an. Fugene 6 gives high efficiency transfection, yet relatively low altered endogenous cellular gene expression 20. Incubate the fugene hd transfection reagentdna mixture for 015 minutes at room temperature. Fugene 6 and fugene hd transfection reagents resulted in the lowest decrease in viability for all three cell types tested with greater than 90% viability figures 24. This is equivalent to over 150 wells in 6 well plate or 35mm tissueculture dishes.
Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Since that time, its popularity has increased due to its ease of use, minimal to no cytotoxicity, and the high level of transfection in many different cell lines. Dualluciferase reporter dlr assay on the spectramax i3x. Aav2 helper free packaging system cell biolabs, inc. Mission lentiviral packaging mix technical bulletin. This manual also presents a simple and rapid transposition method for integrating.
Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. Select the cell line, type of plate used and number of wells to be transfected and a protocol will be. Aav2gfp is produced by cotransfecting 293aav cells cat. Lipofectamine 3000 reagent thermo fisher scientific us. Results show that fugene 6 transfection reagent exhibits very low. Highest transfection efficiency in many cell types and formats e. During development of each reagent, good transfection efficiency was verified in a variety of cells as indicated in the product manual. 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. Refer to the cell lines database for a list of cell types successfully.
We do not recommend the use of other transfection methods, as this leads to reduced luciferase activity from the reporter. 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 hds unique design and composition combined with a simple, fast, user friendly transfection protocol leads to consistent highly reproducible performance, excellent for high throughput application and target validation. Change media to 6 ml of dmem10% fbs 1 hour before transfection. Fugene 6 transfection reagent allows for the efficient delivery of dna vectors from very large plasmids to oligonucleotides and nucleotides into a broad range of cell types with extremely low cytotoxicity. Fugene 6 transfection reagent is stabilized for extended storage. All additional handling steps after the addition of the fugene. Fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. Description part no description piece machine requires piece rubber lip, blue 6.