{"id":289,"date":"2023-07-11T13:16:56","date_gmt":"2023-07-11T20:16:56","guid":{"rendered":"https:\/\/live-usc-dornsife.pantheonsite.io\/pombenet\/?page_id=289"},"modified":"2023-07-27T12:27:04","modified_gmt":"2023-07-27T19:27:04","slug":"telomere-length","status":"publish","type":"page","link":"https:\/\/dornsife.usc.edu\/pombenet\/telomere-length\/","title":{"rendered":"Telomere Length determination"},"content":{"rendered":"\n\n \n \n\n\n\n\n\n\n\n
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Telomere length can vary from strain to strain and is sensitive to a variety of mutants affecting DNA and chromosome function. This protocol is derived from published work by Julie Cooper and modified by Eliana Gomez in our lab.<\/p>\n

 <\/p>\n<\/div>\n<\/div>\n

\n

GEL Protocol<\/h3>\n
\n
    \n
  1. Grow cells for ca. 100 generations (we have done it for approx 50 taking into account that they had already been through several generations before we did the experiment).<\/li>\n
  2. Prepare genomic DNA using standard protocols (see Moreno et al; , or the Nurse Lab Handbook).<\/li>\n
  3. Digest gDNA with EcoRI (ApaI is also used but we’ve had less success with it)<\/li>\n
  4. Run digested DNA on 1 % agarose gel, 1 X TBE.<\/li>\n
  5. Probe with Telomeric oligo probe (5′ GGGTTACAGGTTACAGGTTACA 3′)<\/li>\n<\/ol>\n

     <\/p>\n<\/div>\n<\/div>\n

    \n

    Controls:<\/h3>\n
    \n
      \n
    • WT strains have a smear between 900 and 1100 bp.<\/li>\n
    • taz1<\/em>\u00a0strains have really long telomeres, with the smear very much higher<\/li>\n
    • rhp51<\/em>\u00a0will get slightly longer (above 1100 bp)<\/li>\n
    • rad2<\/em>\u00a0has slightly shorter telomeres.<\/li>\n<\/ul>\n

       <\/p>\n<\/div>\n<\/div>\n

      \n

      Southern Blot using Oligo Probe<\/h3>\n
      \n
        \n
      1. Digest 15ug of genomic DNA overnight.<\/li>\n
      2. Run digested DNA in a 1% agarose 1 x TBE.<\/li>\n
      3. Soak gel in 0.25 N NCl for 10-15 min.<\/li>\n
      4. Rinse gel with dH2O<\/li>\n
      5. Soak gel in 0.4 N NaOH for 30 min<\/li>\n
      6. Wet membrane (I used NEN GeneScreen Plus) with dH2O<\/li>\n
      7. Equilibrate membrane in 0.4 N NaOH for 15 min<\/li>\n
      8. Set up a capillary blot using 0.4 N NaOH (I left transferring for 18 hr)<\/li>\n
      9. Rinse membrane in 2 X SSC<\/li>\n
      10. Cross link DNA to membrane using Stratalinker (you can keep membrane wet at 4\u00b0C in sealed bag till hybridization)<\/li>\n
      11. Prehybridize membrane at 42\u00b0C with approx 10 ml Prehyb solution (make sure that the membrane is completely covered) for a minimum of 2hrs. You should add the DNASSS (Sonicated salmon sperm DNA) once the Prehyb solution reaches 42\u00b0C.<\/li>\n
      12. Before adding the labeled probe change the Prehyb solution for the Hybridization solution. Wait till Hyb Solution reaches to 42\u00b0C and add the labeled oligo. Incubate ON at 42\u00b0C.<\/li>\n
      13. Wash membrane 2 x with 20 ml 6x SSC\/0.1% SDS at room temp.<\/li>\n
      14. I washed membrane again 2 x with 20 ml 6x SSC\/0.1% SDS at 50\u00b0C. Check radioactivity of membrane with Geiger.<\/li>\n<\/ol>\n

         <\/p>\n<\/div>\n<\/div>\n

        \n

        Prehybridizaton solution:<\/h3>\n
        \n
          \n
        • 6x SSC<\/li>\n
        • 5x Denhardt’s<\/li>\n
        • 20 mM NaH2PO4<\/li>\n
        • 500 ug\/ml denatured DNASSS (sonicated salmon sperm DNA), boil for 5 min)<\/li>\n<\/ul>\n

           <\/p>\n<\/div>\n<\/div>\n

          \n

          Hybridization solution:<\/h3>\n
          \n
            \n
          • 6x SSC<\/li>\n
          • 20 mM NaH2PO4<\/li>\n
          • 0.4 % SDS<\/li>\n
          • 300 ug\/ml denatured DNASSS (boil for 5 min)<\/li>\n<\/ul>\n

             <\/p>\n<\/div>\n<\/div>\n

            \n

            Labeling of OLIGO probe:<\/h3>\n
            \n
              \n
            • Telomeric probe: 5′ GGGTTACAGGTTACAGGTTACA<\/li>\n
            • Oligo – 2 ul (100 ng\/ul)<\/li>\n
            • Buffer T4PNK – 5 ul<\/li>\n
            • ATP-gamma32P – 5 ul<\/li>\n
            • PNK – 2 ul<\/li>\n
            • H2O – 36 ul<\/li>\n<\/ul>\n

              Total – 50 ul<\/p>\n

               <\/p>\n

              Incubate at 37*C for 30 min. Stop reaction by adding 5.6 ul of 50 mM EDTA Purify oligo using Bio-Rad Micro Bio-Spin Chromatography columns, Bio-Gel P-6 (Spectrafuge, 1000 xg= 4500 rpm)<\/p>\n<\/div>\n<\/div>\n<\/div>\n\n\n\n<\/div>\n\n\n <\/div><\/div>\n","protected":false},"excerpt":{"rendered":"","protected":false},"author":354,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"page-content-detail.php","meta":{"_acf_changed":false,"footnotes":""},"class_list":["post-289","page","type-page","status-publish","hentry"],"acf":[],"yoast_head":"\nTelomere Length determination - PombeNet Forsburg Lab<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/dornsife.usc.edu\/pombenet\/telomere-length\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Telomere Length determination - 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