Fugene 6 protocol pdf files

Youll notice that these protocols tend to focus around the use of viral methods, which merely reflects the emphasis of my own personal research background. The fugene hd protocol database is a collection of protocols that guide the transfection process for a variety of cell lines. Fugene hd transfection reagent is a nonliposomal formulation designed to. Hill2 cmvs are herpesviruses that establish lifelong latent infection of their hosts. This protocol is used to generate baculoviruses containing gpcr genes of interest for expression in sf9 cells. Four distinct patterns of memory cd8 t cell responses to. Adherent cells can be trypsinized and transfected by the dna. These protocols were developed by promega corporation or fugent, l. Ulkatgfip200 complexes mediate mtor signaling to the.

Using the nanobit ppi system with glomax discover to. Founded in madison, wisconsin in 1995 as fugent co. As for viral production, toxicity will negatively impact the viral yield. After 15 mins incubation at room temperature, the mixture was added to the hek293 cells in a 75 ml flask containing 10 ml dmem medium with 10% fetal bovine serum and 1% antibiotics. The pcmvtag 4a empty vector was used as the negative control. After 12 hr, cells were trypsinized and resuspended in 2. Fugene 6 transfection reagent is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high efficiency and low toxicity. Cells seeded per well 5,000 or 15,000 cells per well 96 well format 3. Detecting ubiquitination of target proteins in live cells. We employ researchers in the areas of organic chemistry, molecular biology, transfection, and chromatography. The simple protocol eliminates tedious culture medium changes and eliminates. Protocols these are provided as microsoft word files. Standard protocol for transient and stable transfection. This protocol is a slight modification of expression systems protocols for generating viruses.

Transfection of sf9 cells in suspension december, 2008 version 1. Cells were harvested 2 d after transfection for coimmunoprecipitation assay or other biochemical or western blot analysis. Here we describe a protocol for detecting and optimizing protacdirected recruitment of a target protein to the psmd3 subunit in the proteasome. The protocol does not require removal of serum or culture. 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. The cells were collected by centrifugation at g for 5 min and then lysed in np40 lysis buffer 25 mm. Like most tcpip protocols, it is based on a clientserver model, with an ftp client on a user machine creating a connection to an ftp server to send and retrieve files to and from the server. In 6cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. Pipette 91 ul of dmem into a sterile polystyrene tube 9. Primary hippocampal neurons from e19 sprague dawley rats were cultured at 150 cellsmm2. The amount of dna and the fugene hd transfection reagent in protocol described bellow calculated to be sufficient to transfect cells in 20. Original article an improved method for increasing the.

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. Gregs retroprep in 293t cells by fugene6 transfection. Original article a combination of vegf165hgf genes is. Two methods for transfection of immortalized mouse embryonic fibroblasts mefs. Sign the compliance checklist and mark each standard compliant, noncompliant or nonapplicable. For using fugene 6 reagent, the above plasmids were mixed with 30 ul fugene 6 transfection reagent. Mix gently by tapping tube and incubate at rt for 15 minutes. Storage and stability fugene 6 reagent is shipped at room temperature. Figure 1 vertiga shaker manufactured by thomson instruments co.

Label the dish initials and place the dish back in the incubator diluting fugene 6 with dmem 7. Incubate the fugene hd transfection reagentdna mixture for 015 minutes at room temperature. Incubate at room temperature for 20 minutes to form complexes. Detecting the interaction of target proteins with e3. Promega offers three premium transfection reagents. Fugene hd transfection reagent is a multicomponent reagent that forms a complex. Fugene hd transfection reagent manual active motif. Additionally, low cell numbers can be transfected in 96well plates.

Everything below should be done in a tissue culture hood, maintaining sterility. Add diluted fugene to tubes of dna dropwise if in duplicate and using 100ul per well, add 200ul to each tube. Welcome to fugent, llc fugent, llc develops and manufactures biotechnology reagents. Its less toxic for cells treatetd with lipo 2000 when you use high cell density 90%. Fugene hd transfection reagent for efficient mammalian. Fugene hd transfection reagent quick protocol fb112pdf. To reduce the scale 1 million 293twell in 6 well plates works well.

Pathogenesis of prostatic small cell carcinoma involves. The cells should be transfected within 510 of adding the chloroquine. 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 6 transfection reagent technical manualpdf 811 kb english. Detecting recruitment of target proteins to the proteasome. I think for virus production capo4 is milder and efficiently enough. Virus titer was determined on lowpassagenumber tzmbl cells by. Add 100ul of transfection mix to each well dropwise and swirl to mix. Assessing protac cell permeability and quantifying vhl. The amount of lgbit plasmid may need to be adjusted based on a the expression level of the. Avoid contact of undiluted fugene hd transfection reagent with the sides of the tube or plate.

While considering a suitable transfection reagent, it is. Biacore peptide binding assay surface plasmon resonance measurements were performed on biacore 3000 biacore at. Fugene 6 transfection reagent is a proprietary blend of lipids and other components. I did a midiprep from qiagen and checked using the nanodrop. Fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. Although a young company, fugent has revolutionized biological study with its. Fugene 6 transfection reagent is gentle on the cells. Plate cells so they will be 6080% confluent at the time of transfection. Fugene hd transfection reagent quick protocol fb112pdf 99 kb english.

For specific goclone transfection protocol examples, see our online resources page. Fugene hd transfection reagent promega corporation. Pipette 9 ul of fugene 6 directly into the dmem avoid direct contact between fugene and the tube wall 10. The main objectives of ftp were to make file transfer simple. A tm total rna kit omega biotek as instructed by the manufacturer. Kinetically detecting and quantitating protacinduced. For selection of cells in which piggybac transposed, several 400 ml aliquots of cells were plated into one 10cm dish with 10 ml media containing g418 700 mg ml, resulting in.

Prepare a 5x concentration of test protac titration. According to the attached fugene protocol, its recomended to use 10ug for a 10cm petridish reaction. One the day before transfection, split 293t cells 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. But avoid asking for help, clarification, or responding to other answers. Transfect 293t cells with plasmids using fugene 6 1. Add 22 l of 25mm chloroquine to each 15cm dish for 10cm plate 7. Fugene 6 transfection reagent is stabilized for extended storage. 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. It is helpful to list documents in the order they appear in the file. This assay is based on nanobret technology, a proximitybased method dependent upon energy transfer from a. File folders must include completed documentation for the three years of.

Calling cards for dnabinding proteins in mammalian cells. Fugene 6 transfection reagent protocol promega corporation. Although to the best of my knowledge these protocols are well characterized and thoroughly proofread, theres no guarantee provided. 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. Transfection of immortalized mefs emory university. Four distinct patterns of memory cd8 t cell responses to chronic murine cytomegalovirus infection1 michael w. List primaryprotocol and secondaryprocess indicator documents on the compliance checklist. Always mix fugene 6 reagent prior to use vortex for one second or use inversion. Description fugene 6 transfection reagenta is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high e.

Immunoprecipitation and immunoblotting were performed as described previously 10. Thanks for contributing an answer to stack overflow. Aseptically place fugene 6 and gfpactin dna in the hood 8. Fix protocol specification provides format for electronic messages and communication model fix can be used by financial institutions like brokerdealers, exchanges, institutional investors and others in the industry to communicate among each other it is widely used protocol in. Vwr offers transfection reagents for numerous applications, including bioproduction, in vivo work, viral production, dna and sirna transfection, crisprcas 9 and more. Freeze 48hr plate, read plates additional materials needed. Immunoblot analysis cells were washed with pbs and lysed in ripa buffer 50 mmtris,ph7. Lipofectamine and pei this protocol is from the kahn lab, optimized for mefs being transfected in 6well plates, with thanks to laura newman and rachel turn.

Before the experiment, i place the optimem at room temperature for 10min and vortex the fugene 6 to make it evenly. The protocol does not require removal of serum or culture medium and does not. Three microliters of fugene 6 transfection reagent is combined with 12 g of reportergene vector dna, and used to transfect cos1 cells. Prepare a transfection mixture consisting of the dna amounts listed in table 1. Transient transfection of 293t cells faculty websites. Transfection of sf9 cells in suspension nih common fund. For 96well format, seed cells in 100ul total volume per well. Lentiviral preparation and viral infection lentiviral shrna transduction was performed as described previously. Fugene 6 reagent complex prior to plating, making it a strong candidate for high throughput applications.