{"id":747,"date":"2013-09-06T16:55:56","date_gmt":"2013-09-06T15:55:56","guid":{"rendered":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?page_id=747"},"modified":"2014-11-26T11:30:03","modified_gmt":"2014-11-26T10:30:03","slug":"page-separation-of-small-rnas","status":"publish","type":"page","link":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/","title":{"rendered":"PAGE separation of small RNAs"},"content":{"rendered":"

For the resolution of RNAs between 15 and 500 nucleotides in length, depending on the percentage acrylamide used.<\/p>\n

Materials<\/em><\/strong><\/p>\n

Formamide buffer (0.01M EDTA, 0.1% bromophenol blue, deionised formamide)<\/p>\n

Gel set-up<\/strong><\/em><\/p>\n

\"PAGE<\/a>NB: bromophenol blue migration and run time based on an 8×5 cm mini-gel<\/i><\/p>\n

    \n
  1. Put together the running assembly, fill with 0.5x TBE, remove the comb and clean the wells with running buffer<\/li>\n
  2. Pre-run the gel for 1 hour at 300 V<\/li>\n<\/ol>\n

    Sample preparation & gel running<\/i><\/b><\/p>\n

      \n
    1. Resuspend 1 \u03bcg RNA in 2.5 \u03bcl ddH2<\/sub>O (RNAse-free) and add 7.5 \u03bcl formamide buffer<\/strong><\/li>\n
    2. Denature at 65\u00b0C for 15 minutes, spin briefly, place on ice; load samples taking care not to overflow into neighbouring wells<\/li>\n
    3. Run gel at 300 V or until the dye front runs off the gel<\/li>\n<\/ol>\n

      Staining & transfer<\/i><\/b><\/p>\n

        \n
      1. Remove the gel from the assembly and place in 0.5x TBE containing 50ng\/ml ethidium bromide for 10 minutes; de-stain in 0.5x TBE for 10 minutes<\/li>\n
      2. Transfer to positively charged nylon membrane (e.g. Zeta membrane, Bio-Rad) using a semi-dry electroblotter (e.g. transblotter, Bio-Rad)<\/li>\n
      3. Pre-soak the membrane and two pieces of extra thick filter paper in 0.5x TBE for 10 minutes<\/li>\n
      4. Set-up the transfer stack (bottom: filter paper, membrane, gel, filter paper: top) and transfer for 1 hour at 250 mA<\/li>\n
      5. Cross-link in a Stratalinker or similar<\/li>\n<\/ol>\n

        Pre-hybridise and hybridise as per standard northern protocol<\/p>\n","protected":false},"excerpt":{"rendered":"

        For the resolution of RNAs between 15 and 500 nucleotides in length, depending on the…<\/p>\n","protected":false},"author":116,"featured_media":0,"parent":17,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-747","page","type-page","status-publish","hentry","odd"],"yoast_head":"\nPAGE separation of small RNAs - Alsford 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:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"PAGE separation of small RNAs - Alsford Lab\" \/>\n<meta property=\"og:description\" content=\"For the resolution of RNAs between 15 and 500 nucleotides in length, depending on the...\" \/>\n<meta property=\"og:url\" content=\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/\" \/>\n<meta property=\"og:site_name\" content=\"Alsford Lab\" \/>\n<meta property=\"article:modified_time\" content=\"2014-11-26T10:30:03+00:00\" \/>\n<meta property=\"og:image\" content=\"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/files\/2013\/09\/PAGE-small-RNAs-table.png\" \/>\n<meta name=\"twitter:label1\" content=\"Estimated reading time\" \/>\n\t<meta name=\"twitter:data1\" content=\"1 minute\" \/>\n<script type=\"application\/ld+json\" class=\"yoast-schema-graph\">{\"@context\":\"https:\/\/schema.org\",\"@graph\":[{\"@type\":\"WebPage\",\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/\",\"url\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/\",\"name\":\"PAGE separation of small RNAs - Alsford Lab\",\"isPartOf\":{\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/#website\"},\"primaryImageOfPage\":{\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#primaryimage\"},\"image\":{\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#primaryimage\"},\"thumbnailUrl\":\"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/files\/2013\/09\/PAGE-small-RNAs-table.png\",\"datePublished\":\"2013-09-06T15:55:56+00:00\",\"dateModified\":\"2014-11-26T10:30:03+00:00\",\"breadcrumb\":{\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#breadcrumb\"},\"inLanguage\":\"en-GB\",\"potentialAction\":[{\"@type\":\"ReadAction\",\"target\":[\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/\"]}]},{\"@type\":\"ImageObject\",\"inLanguage\":\"en-GB\",\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#primaryimage\",\"url\":\"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/files\/2013\/09\/PAGE-small-RNAs-table.png\",\"contentUrl\":\"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/files\/2013\/09\/PAGE-small-RNAs-table.png\"},{\"@type\":\"BreadcrumbList\",\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#breadcrumb\",\"itemListElement\":[{\"@type\":\"ListItem\",\"position\":1,\"name\":\"Protocols\",\"item\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/\"},{\"@type\":\"ListItem\",\"position\":2,\"name\":\"PAGE separation of small RNAs\"}]},{\"@type\":\"WebSite\",\"@id\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/#website\",\"url\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/\",\"name\":\"Alsford Lab\",\"description\":\"Trypanosome molecular and cellular biology\",\"potentialAction\":[{\"@type\":\"SearchAction\",\"target\":{\"@type\":\"EntryPoint\",\"urlTemplate\":\"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?s={search_term_string}\"},\"query-input\":{\"@type\":\"PropertyValueSpecification\",\"valueRequired\":true,\"valueName\":\"search_term_string\"}}],\"inLanguage\":\"en-GB\"}]}<\/script>\n<!-- \/ Yoast SEO plugin. -->","yoast_head_json":{"title":"PAGE separation of small RNAs - Alsford Lab","robots":{"index":"index","follow":"follow","max-snippet":"max-snippet:-1","max-image-preview":"max-image-preview:large","max-video-preview":"max-video-preview:-1"},"canonical":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/","og_locale":"en_GB","og_type":"article","og_title":"PAGE separation of small RNAs - Alsford Lab","og_description":"For the resolution of RNAs between 15 and 500 nucleotides in length, depending on the...","og_url":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/","og_site_name":"Alsford Lab","article_modified_time":"2014-11-26T10:30:03+00:00","og_image":[{"url":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/files\/2013\/09\/PAGE-small-RNAs-table.png","type":"","width":"","height":""}],"twitter_misc":{"Estimated reading time":"1 minute"},"schema":{"@context":"https:\/\/schema.org","@graph":[{"@type":"WebPage","@id":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/","url":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/","name":"PAGE separation of small RNAs - Alsford Lab","isPartOf":{"@id":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/#website"},"primaryImageOfPage":{"@id":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#primaryimage"},"image":{"@id":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#primaryimage"},"thumbnailUrl":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/files\/2013\/09\/PAGE-small-RNAs-table.png","datePublished":"2013-09-06T15:55:56+00:00","dateModified":"2014-11-26T10:30:03+00:00","breadcrumb":{"@id":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#breadcrumb"},"inLanguage":"en-GB","potentialAction":[{"@type":"ReadAction","target":["https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/"]}]},{"@type":"ImageObject","inLanguage":"en-GB","@id":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#primaryimage","url":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/files\/2013\/09\/PAGE-small-RNAs-table.png","contentUrl":"http:\/\/blogs.lshtm.ac.uk\/alsfordlab\/files\/2013\/09\/PAGE-small-RNAs-table.png"},{"@type":"BreadcrumbList","@id":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/page-separation-of-small-rnas\/#breadcrumb","itemListElement":[{"@type":"ListItem","position":1,"name":"Protocols","item":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/protocols\/"},{"@type":"ListItem","position":2,"name":"PAGE separation of small RNAs"}]},{"@type":"WebSite","@id":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/#website","url":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/","name":"Alsford Lab","description":"Trypanosome molecular and cellular biology","potentialAction":[{"@type":"SearchAction","target":{"@type":"EntryPoint","urlTemplate":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/?s={search_term_string}"},"query-input":{"@type":"PropertyValueSpecification","valueRequired":true,"valueName":"search_term_string"}}],"inLanguage":"en-GB"}]}},"_links":{"self":[{"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/pages\/747","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/users\/116"}],"replies":[{"embeddable":true,"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/comments?post=747"}],"version-history":[{"count":6,"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/pages\/747\/revisions"}],"predecessor-version":[{"id":915,"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/pages\/747\/revisions\/915"}],"up":[{"embeddable":true,"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/pages\/17"}],"wp:attachment":[{"href":"https:\/\/blogs.lshtm.ac.uk\/alsfordlab\/wp-json\/wp\/v2\/media?parent=747"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}