It can be used in the presence or absence of serum, and its. The protocol can be scaled to produce different amounts of virus as needed. The protocol does not require removal of serum or culture. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Fugene 6 transfection reagent protocol promega corporation. To reduce the scale 1 million 293twell in 6 well plates works well. Fugene hd transfection reagent manual active motif.
Fugene 6 is a multicomponent lipidbased transfection reagent that complexes with and transports. The cells are ready for transfection after 1820 hours, or when they are. Quick vortex, tap tube to settle, and sit 5 minutes at rt. Overview protocols specifications resources your everyday transfection reagent fugene 6 transfection reagent provides sturdy results in a wide variety of standard laboratory cell lines, making it ideal for everyday use. Gregs retroprep in 293t cells by fugene6 transfection. Comparison of transfection reagent performance in hek293 cells. This protocol is for transfection to produce pseudotyped lentivirus using fugene 6 transfection reagent in a 10cm dish. This reagent combines high transfection efficiency with exceptionally low cytotoxicity. Cas 64175 fugene 6 transfection reagent, 1814443 ethanol msds toxicity property. A protocol for the fugene 6 transfection reagent, a nonliposomal reagent that transfects dna into a wide variety of cell lines with high. Recommended transfection protocol for initial transfection experiment and transfection optimization we suggest using 96well plate format. Formulation fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. Fugene 6 transfection reagent is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high efficiency and low toxicity. Fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials.
The day before transfection plate adherent cells in the amount that by the time of transfection. Im also curious about this, cause the rnaimax leads too much cell death in hk2 cells cultured with ksfm, fugene hd is low toxicity and high efficiency, so if i can delivery sirna fugene, i could. Lipofectamine rnaimax reagent thermo fisher scientific. Fugene hd transfection reagent quick protocol fb112pdf. Fugene 6 transfection reagent is gentle on the cells. Transfect cells according to the manufacturers protocol. Fugene 6 transfection reagent is a nonliposomal, multicomponent reagent proven to efficiently transfect more than 700 cell types. This is the protocol that i use when doing transfections with fugene 6. Fugene 6 transfection reagent technical manualpdf 811 kb english. Production of retroviruses using fugene6 32100 1 on the day before transfection, plate 0. To a sterile tube or u or vbottom plate, add 90 98l of medium prewarmed to room temperature so that the final volume after. 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.
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. Storage and stability fugene 6 reagent is shipped at room temperature. 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. Volume of dme serumfree medium and fugene 6 transfection reagent required for various formats format dme serumfree medium fugene 6 transfection reagent 96 well plate 9615 lwell0. Before the experiment, i place the optimem at room temperature for 10min and vortex the fugene 6 to make it evenly.
Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. For your convenience, the essential components of this protocol are now available in the gibco liver cancer starter kit. The simple protocol eliminates tedious culture medium changes and eliminates. The use of fugene hd does not require culture medium change or serum removal during transfection experiment. Follow the transfection reagent protocol for seeding cells. For specific goclone transfection protocol examples, see our online resources page. Fugene 6 transfection reagent has been commercially available since 1997. Production of retroviruses using fugene 6 32100 1 on the day before transfection, plate 0.
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. In 6 cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. Fugene 6 transfection reagent is a nonliposomal formulation designed to transfect. Fugene 6 transfection reagent university of pittsburgh. During development of each reagent, good transfection efficiency was verified in a variety of cells as indicated in the product manual.
The protocol does not require removal of serum or culture medium and does not. Fugene 6 transfection reagenta is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high e. The fugene 6 transfection reagent provides high expression levels with minimal impact on cell health. Test a variety of ul fugene hd to ug dna ratios ranging from 2. Apr 20, 2006 fugene 6 transfection reagent is a nonliposomal, multicomponent reagent proven to efficiently transfect more than 700 cell types. Minimizing reagentdependent side effects as analyzed by geneexpression profiling and cytotoxicity assays article pdf available in nature methods 35 april. Plate cells so they will be 6080% confluent at the time of transfection. 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. 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.
Mission lentiviral packaging mix technical bulletin. Citation list of established cells transfected with fugene 6. Fugene hd transfection reagent lets you work in your cancer model. Fugene 6 transfection reagent is stabilized for extended storage. Standard protocol for transient and stable transfection. It may also refer to other methods and cell types, although other terms are often preferred. Description fugene 6 transfection reagenta is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high e. Tips and tricks on how to improve transfection experiments. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Msds fugene 6 transfection reagent, 1814443 ethanol. The database includes over 25 cancer models and provides transfection protocols specific for each cell line. Fugener hd transfection reagent quick protocol card, fb112. Overview of adherent cell transfection protocol for a 96well plate. Fugene is a transfection reagent that apparently increases transfection rates for animal cells.
Protocol 2 producing lentivirus in hek293t cells using a 2. Lipofectamine 3000 transfection reagent hepg2 liver. In 6cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. The simple protocol eliminates tedious culture medium changes and eliminates a source of variation. The amount of dna and the fugene hd transfection reagent in protocol. Citation list of established cells transfected with fugene 6 transfection reagent 10t12 timothy a. Fugene 6 transfection reagent provides sturdy results in a wide variety of standard laboratory cell lines, making it ideal for everyday use. Use the fugene hd protocol database to see if transfection conditions for your cell line are available. Choosing a transfection reagent is easier when you can rely on the work of researchers who have used it before you.
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