![]() Separate the RNA-protein complexes from free RNA using gel electrophoresis and membrane transfer. Proteinase K digestion is then performed in order to remove protein from the complexes. This step leaves a peptide at the cross-link site, allowing for the identification of the cross-linked nucleotide. After ligating RNA linkers to the 5' ends, cDNA is synthesized by RT-PCR. The last cDNA nucleotide is identified by high-throughput sequencing. Mapping the reads back to the transcriptome can identify the interaction sites. Harvest brain, spinal cord, or other target tissues from mice. Sit the tissue in ice-cold HBSS until the harvest is complete. Set up a 500mL Stericup, remove the cellulose filter, and make a conical filter out of a sheet of 200μm nylon mesh to replace it. ![]() The resulting cell suspension is about 100mL for 10 brains. Next, transfer the tissue suspension to 50mL tubes and spin at 2500g for 5min at 4☌. ![]() Remove the supernatant and resuspend the tissue in approximate 10 times the original volume of tissue. Place the suspension in a 150mm tissue culture dish and irradiate the suspension for 400mJ/cm 2. Use a dish of ice underneath the tissue suspension as you crosslink to keep the suspension cold.Ĭollect the irradiated suspension in a 50mL tube, then wash out each plate with an additional 5mL of HBSS, also collecting this wash. Spin down the tissue again at 2500rpm for 5min at 4☌. Resuspend the tissue in 2 times volume of the original tissue volume and pipet the solution into tubes. Spin the tubes briefly and take off the supernatant. Pipet 100μL of protein A Dynabead beads solution. Place beads in magnetic stand to capture and wash beads with 500μL of 1X PXL. Resuspend beads in 100μL of 1X PXL and add an appropriate amount of your anti-RNA binding protein antibody. Rock the tube for 30min to 45min at room temperature to bind the antibody to the beads. Lyse each 100μL of cross-linked cells using 100μL of ice-cold 1X PXL. Add 10μL RNasin and 10μL RQ1 DNase to each tube incubate at 37☌ for 15min. Add 1μL RNase T1 stock (1 U/μL) to the solution incubate at 37☌ for10 min, shaking at 1000rpm. Next, spin the lysates in a prechilled micro-ultracentrifuge at 90K for 25min at 4☌.įor immunoprecipitation, carefully remove the supernatant from the pelleted debris and add supernatant to one prepared tube of beads. Use about 100μL of beads per 300μL of cross-linked tissue. For more information on how to obtain warranty service, write, e-mail or telephone ProClip at 4915 Voges Rd., Madison, WI 53718 USA.Wash three times with 1mL ice-cold 1X PXL and twice with 1mL ice-cold 1X PNK+. You will bear all shipping, packaging, and all other costs, excluding labor and parts, necessary to effectuate repair, replacement or refund under this warranty. If shipped to ProClip, the buyer must first call ProClip at +1 80 or e-mail ProClip at to obtain a return approval and return number. Warranty service is available to you by delivering the Product during the warranty period to the company it was purchased from or to ProClip at 4915 Voges Rd., Madison, WI 53718 USA and providing proof of purchase, price and date. All replaced Products shall become the property of ProClip. Repair or replacement parts or Products will be furnished on an exchange basis and will either be new or reconditioned. If any Product should become defective within the warranty period, ProClip, at its option, will replace or repair it. ProClip warrants that the Products will be free of defects in materials and workmanship for a period of one (1) year from the date of purchase.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |