Profiling the expression pattern of gpi transamidase. Adherent cells can be trypsinized and transfected by the dna. Before the experiment, i place the optimem at room temperature for 10min and vortex the fugene 6 to make it evenly. An example showing optimization of transfection conditions for the fugene hd reagent in a 96well plate is shown in figure 2. Results show that fugene 6 transfection reagent exhibits very low. However, after overnight incubation, almost 90% of cells are floating in the medium which are supposed to be dead cells. Stable cell line generation with pcoron1002egfpc1psld 32 7. In 6 cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. With fugene hd, there is no need to change media after an. Dna ratio, add 6l of fugene 6 transfection reagent to the medium, and mix immediately add fugene 6 transfection reagent directly to medium.
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. 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. Production of retroviruses using fugene6 32100 1 on the day before transfection, plate 0. Vwr offers transfection reagents for numerous applications, including bioproduction, in vivo work, viral production, dna and sirna transfection, crisprcas 9 and more. 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. Additional items required items required but not provided transfection the procedure is modified for using fugene 6 roche in a 12well culture plate. 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. Plate slightly more cells when making vsvg pseudotyped viruses. All additional handling steps after the addition of the fugene. 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.
While considering a suitable transfection reagent, it is. Product manual aav2 helper free packaging system catalog number. The cells are ready for transfection after 1820 hours, or when they are about 2530% confluent. Thank you so muchmcri tried both of them and lipofectamine 2000 was much betterin 293ft cells. Aav2 helper free packaging system cell biolabs, inc. 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. Cell biolabs aav helperfree system allows the production of infectious recombinant human adeno. Promega fugene 6 transfection reagent fugene 6 transfection reagent is a nonliposomal formulation designed to transfect plasmid dna into a wide. Incubate the fugene hd transfection reagentdna mixture for 015 minutes at room temperature. Fugene 6 transfection reagent is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high efficiency and low toxicity.
Many signal transduction events and transcription factors have been demonstrated to be associated with er stress. Guidelines for transfecting luciferase reporter vector with fugene 6. The allinone system for the inducible expression of shrna. All additional handling steps after the addition of the fugene 6 reagent. Lipofectamine 3000 reagent thermo fisher scientific us. To be the most responsive supplier of biological reagents and reagent systems used in research and applied technology applications worldwide. 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. The fugene hd transfection reagent is lipidbased, simpletouse and can yield high transfection efficiencies with minimal cytotoxicity. This is equivalent to over 150 wells in 6 well plate or 35mm tissueculture dishes.
Hi,guys, currently im doing sirna transfection to hela cells with fugene 6. A protocol for the fugene 6 transfection reagent, a nonliposomal reagent that transfects dna into a wide variety of cell. The fugene hd transfection reagent protocol database has protocols for transfecting your cell line. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Change media to 6 ml of dmem10% fbs 1 hour before transfection.
Do not allow undiluted fugene 6 transfection reagent to contact the sides of the tube or u or vbottom plate. Fugene 6 transfection reagent is gentle on the cells. Fugene is apparently a nonliposomal formulation of blended lipids, but its actual contents are not revealed anywhere. Obtain optimal results with highsensitivity quantiation of luciferase activity with 6decade linear range. Find msds or sds, a coa, data sheets and more information. Karcher br 5560 w ep instructions manual pdf download. Fugene 6 reagent complex prior to plating, making it a strong candidate for high throughput applications. 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. Highest transfection efficiency in many cell types and formats e. Select the cell line, type of plate used and number of wells to be transfected and a protocol will be. 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. 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.
Citation list of established cells transfected 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. All in vivo work with gl261 glioma cells was done in c57bl6 wildtype mice charles river laboratories, wilmington, ma, usa. This reagent is suitable for use in media with or without serum, and for transient or stable transfection. Storage and stability fugene 6 reagent is shipped at room temperature. Gene promoter reporter vector user manual page 5 materials materials provided sequence information is available on our website. In transfections, skbr3 cells were transfected with pmeebhapigs and pmeebgstgpi8 plasmids in the presence of the fugene 6 transfection regent. Lipofectamine 2000 transfection reagent was the most toxic, and produced as high as a 55% drop in viability in hek293 cells figure 2.
Improving the success of your transfection promega. Altered activity patterns of transcription factors induced. Citation list of established cells transfected with fugene. The following figure demonstrates typical results seen with cell biolabs aav helperfree system. Production of retroviruses using fugene 6 32100 1 on the day before transfection, plate 0. Fugene 6 transfection reagent technical manual promega.
In animal cells, transfection is the preferred term as. Apr 20, 2006 fugene 6 gives high efficiency transfection, yet relatively low altered endogenous cellular gene expression 20. Always mix fugene 6 reagent prior to use vortex for one second or use inversion. Aav2gfp is produced by cotransfecting 293aav cells cat. Fugene 6 transfection reagent technical manualpdf 811 kb english. These transfer vectors are accompanied by modified recipient baculovirus dna that has been engineered towards improved protein production. It may also refer to other methods and cell types, although other terms are often preferred.
Fugene 6 transfection reagent has been commercially available since 1997. Citation list of established cells transfected with fugene 6 transfection reagent 10t12 timothy a. Refer to the cell lines database for a list of cell types successfully. This manual also presents a simple and rapid transposition method for integrating. Transfection the procedure is modified for using fugene 6 roche in a 12well culture plate. E to create a plate with titrations and replicates of different transfection conditions. Add 36 ul of fugene directly into 600 l of optimem. Mission lentiviral packaging mix technical bulletin. 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. Prepare transfection mixture of plasmid dna and fugene transfection reagent roche according to manufacturers instructions. Reagent wst1 roche applied science according to the instructions. Fugener hd transfection reagent technical manual tm328. Fugene hd transfection reagent manual active motif.
Fugene 6 transfection reagent is a nonliposomal formulation designed to. Then the 293 cells were infected with ad5 moi 10 for helper function the cells. 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. 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. Avoid contact of undiluted fugene hd transfection reagent with the sides of the tube or plate. Additionally, low cell numbers can be transfected in 96well plates. Study gene expression using the dualluficerase reporter dlrassay on the spectramax i3x multimode microplate reader with spectramax injector cartridge.
Dualluciferase reporter dlr assay on the spectramax i3x. Olson 2000 activation of the myocyte enhancer factor2 transcription factor by calciumcalmodulindependent protein kinasestimulated binding of 1433 to histone deacetylase 5. Improving the success of your transfection promega connections. We do not recommend the use of other transfection methods, as this leads to reduced luciferase activity from the reporter. Promega corporation is a worldwide leader in applying biochemistry and molecular biology to the development of innovative, highvalue products for the life sciences. Lipofectamine 2000 was the most toxic reagent, with the highest cell death in the hek293 and hep g2 cell lines 55% loss in viability. 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. During development of each reagent, good transfection efficiency was verified in a variety of cells as indicated in the product manual. Fugene 6 transfection reagent is stabilized for extended storage. Description part no description piece machine requires piece rubber lip, blue 6. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells.
The process in which er stress affects or interacts with other pathways is. This manual introduces a set of novel baculovirus transfer vectors that specifically enable multigene applications trowitzsch et al. Lipofectamine2000 works quite well in 293cells 90% confluent in my. Gently shake the culture plate several times and harvest all media, including cells, in a sterile tube. Fugene 6 transfection reagent has to be prepared in combination with serumfree dme medium. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. The use of fugene hd does not require culture medium change or serum removal during. 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 a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. Introduction to the design u0026 analysis of algorithms solution manual. In 6cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. Fugene 6 gives high efficiency transfection, yet relatively low altered endogenous cellular gene expression 20.
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