Detecting ubiquitination of target proteins in live cells. Standard protocol for transient and stable transfection. Add diluted fugene to tubes of dna dropwise if in duplicate and using 100ul per well, add 200ul to each tube. This protocol is used to generate baculoviruses containing gpcr genes of interest for expression in sf9 cells. L always mix fugene 6 transfection reagent prior to use vortes. Label the dish initials and place the dish back in the incubator diluting fugene 6 with dmem 7. All three offer broad spectrum transfection capability and low.
Ulkatgfip200 complexes mediate mtor signaling to the. File folders must include completed documentation for the three years of. The fugene hd protocol database is a collection of protocols that guide the transfection process for a variety of cell lines. The pcmvtag 4a empty vector was used as the negative control. The protocol does not require removal of serum or culture medium and does not. Pathogenesis of prostatic small cell carcinoma involves.
One the day before transfection, split 293t cells 0. Aseptically place fugene 6 and gfpactin dna in the hood 8. Incubate the fugene hd transfection reagentdna mixture for 015 minutes at room temperature. Fugene hd transfection reagent for efficient mammalian. Fugene 6 reagent complex prior to plating, making it a strong candidate for high throughput applications. Everything below should be done in a tissue culture hood, maintaining sterility.
Although to the best of my knowledge these protocols are well characterized and thoroughly proofread, theres no guarantee provided. This assay is based on nanobret technology, a proximitybased method dependent upon energy transfer from a. Founded in madison, wisconsin in 1995 as fugent co. Fugene 6 transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, sterilefiltered, and packaged in glass vials. Immunoblot analysis cells were washed with pbs and lysed in ripa buffer 50 mmtris,ph7. Vwr offers transfection reagents for numerous applications, including bioproduction, in vivo work, viral production, dna and sirna transfection, crisprcas 9 and more. Additionally, low cell numbers can be transfected in 96well plates.
Assessing protac cell permeability and quantifying vhl. Original article an improved method for increasing the. The cells should be transfected within 510 of adding the chloroquine. Promega offers three premium transfection reagents. It is helpful to list documents in the order they appear in the file. 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. Storage and stability fugene 6 reagent is shipped at room temperature. For specific goclone transfection protocol examples, see our online resources page. Fugene 6 transfection reagent technical manualpdf 811 kb english. Cells seeded per well 5,000 or 15,000 cells per well 96 well format 3. Before the experiment, i place the optimem at room temperature for 10min and vortex the fugene 6 to make it evenly. 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.
Figure 1 vertiga shaker manufactured by thomson instruments co. Protocols these are provided as microsoft word files. Cells were harvested 2 d after transfection for coimmunoprecipitation assay or other biochemical or western blot analysis. Transient transfection of 293t cells faculty websites.
Fugene hd transfection reagent quick protocol fb112pdf 99 kb english. Avoid contact of undiluted fugene hd transfection reagent with the sides of the tube or plate. Fugene hd transfection reagent promega corporation. Transfection of sf9 cells in suspension nih common fund. Pipette 9 ul of fugene 6 directly into the dmem avoid direct contact between fugene and the tube wall 10. Transfection of immortalized mefs emory university. Fugene 6 transfection reagent is stabilized for extended storage. While considering a suitable transfection reagent, it is. The main objectives of ftp were to make file transfer simple. Plate cells so they will be 6080% confluent at the time of transfection. Immunoprecipitation and immunoblotting were performed as described previously 10. Fugene 6 transfection reagent is gentle on the cells. Calling cards for dnabinding proteins in mammalian cells.
Biacore peptide binding assay surface plasmon resonance measurements were performed on biacore 3000 biacore at. Three microliters of fugene 6 transfection reagent is combined with 12 g of reportergene vector dna, and used to transfect cos1 cells. For using fugene 6 reagent, the above plasmids were mixed with 30 ul fugene 6 transfection reagent. These protocols were developed by promega corporation or fugent, l. Fugene hd transfection reagent is a multicomponent reagent that forms a complex. Detecting recruitment of target proteins to the proteasome.
According to the attached fugene protocol, its recomended to use 10ug for a 10cm petridish reaction. In 6cmdish, when the confluence of 293t reachs 50%,i conduct the transfection. Fugene hd transfection reagent quick protocol fb112pdf. Add 100ul of transfection mix to each well dropwise and swirl to mix. The cells were collected by centrifugation at g for 5 min and then lysed in np40 lysis buffer 25 mm.
Add 22 l of 25mm chloroquine to each 15cm dish for 10cm plate 7. Description fugene 6 transfection reagenta is a nonliposomal reagent that transfects dna into a wide variety of cell lines with high e. Four distinct patterns of memory cd8 t cell responses to. Always mix fugene 6 reagent prior to use vortex for one second or use inversion. Although a young company, fugent has revolutionized biological study with its.
The amount of lgbit plasmid may need to be adjusted based on a the expression level of the. 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. After 12 hr, cells were trypsinized and resuspended in 2. Hill2 cmvs are herpesviruses that establish lifelong latent infection of their hosts. Four distinct patterns of memory cd8 t cell responses to chronic murine cytomegalovirus infection1 michael w. 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. Original article a combination of vegf165hgf genes is.
Fugene 6 transfection reagent is a proprietary blend of lipids and other components. 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. I think for virus production capo4 is milder and efficiently enough. 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. Kinetically detecting and quantitating protacinduced. A tm total rna kit omega biotek as instructed by the manufacturer. Mix gently by tapping tube and incubate at rt for 15 minutes. Prepare a 5x concentration of test protac titration. 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. I did a midiprep from qiagen and checked using the nanodrop. Fugene 6 transfection reagent protocol promega corporation. Freeze 48hr plate, read plates additional materials needed. Optimize the transfection protocol for your specific cell line and experimental design using the lightswitch transfection optimization kit tfxopt.
Primary hippocampal neurons from e19 sprague dawley rats were cultured at 150 cellsmm2. The protocol does not require removal of serum or culture. Thanks for contributing an answer to stack overflow. Two methods for transfection of immortalized mouse embryonic fibroblasts mefs. Transfect 293t cells with plasmids using fugene 6 1. Sign the compliance checklist and mark each standard compliant, noncompliant or nonapplicable. Welcome to fugent, llc fugent, llc develops and manufactures biotechnology reagents. Transfection of sf9 cells in suspension december, 2008 version 1. Prepare a transfection mixture consisting of the dna amounts listed in table 1. 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 hd transfection reagent manual active motif. For 96well format, seed cells in 100ul total volume per well. Using the nanobit ppi system with glomax discover to.
The simple protocol eliminates tedious culture medium changes and eliminates. List primaryprotocol and secondaryprocess indicator documents on the compliance checklist. Incubate at room temperature for 20 minutes to form complexes. This protocol is a slight modification of expression systems protocols for generating viruses. To reduce the scale 1 million 293twell in 6 well plates works well. But avoid asking for help, clarification, or responding to other answers. 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. 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. Lentiviral preparation and viral infection lentiviral shrna transduction was performed as described previously. Its less toxic for cells treatetd with lipo 2000 when you use high cell density 90%. Here we describe a protocol for detecting and optimizing protacdirected recruitment of a target protein to the psmd3 subunit in the proteasome. We employ researchers in the areas of organic chemistry, molecular biology, transfection, and chromatography. Gregs retroprep in 293t cells by fugene6 transfection. 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.153 552 36 1328 186 1231 142 1403 939 120 1217 1099 1497 704 397 1138 1159 1130 1307 557 1356 2 814 561 433 1375 506 1269 1218 565 345 1209 1109 401 586 1214 889 7 974 1291 1215 1310 393 836 1158 197 567