{"id":228,"date":"2012-04-10T16:04:13","date_gmt":"2012-04-10T16:04:13","guid":{"rendered":"https:\/\/wordpress.clarku.edu\/debrobertson\/?page_id=228"},"modified":"2012-05-23T17:19:54","modified_gmt":"2012-05-23T17:19:54","slug":"rna-extraction-from-red-algae","status":"publish","type":"page","link":"https:\/\/wordpress.clarku.edu\/debrobertson\/laboratory-protocols\/rna-extraction-from-red-algae\/","title":{"rendered":"RNA Extraction from Red Algae"},"content":{"rendered":"<p align=\"center\"><em>(Contributed by S. Ghoshroy, edited 11\/15\/2011 by DLR)<\/em><\/p>\n<h2>Preparation of material:<\/h2>\n<p>Read the following set of instructions before you plan to extract RNA.\u00a0 Solutions, and glassware should be ready before you take the samples out of the freezer for extraction.\u00a0 Before preparing solutions or starting extractions, metal scoops and spatulas, a mortar and pestle, a few beakers, Erlenmeyer flasks, and bottles should be baked at 275\u00b0 C for 2 to 3 h.\u00a0 Treating solutions and water with 0.1% diethyl pyrocarbonate (DEPC) can reduce RNAse contamination.\u00a0 Compounds containing amine groups (e.g. Tris) cannot be DEPC and must be made using ultrapure stocks and DEPC water. Plasticware (blue (100-1000\u03bcl) and white (20-200\u03bcl) pipette tips, preloaded boxes, test tubes, etc.) untouched by humans should be autoclaved.<\/p>\n<p>DEPC-treated water can be generated by treatment with 0.1% DEPC for 1 hour at 37\u00baC and autoclaving for 15-20 at 15 psi. For more information about working with RNA, we recommend reading &#8220;How to win the battle with RNase&#8221; in Sambrook and Russell, Molecular Cloning, Volume 1. 782-7.85.<\/p>\n<p>&nbsp;<\/p>\n<h2>Preparation of Solutions.<\/h2>\n<ol start=\"1\">\n<li>Prepare 70% EtOH for RNA use with DEPC-treated water in RNase-free bottle or centrifuge tube.<\/li>\n<li>Preparation of solution D (denaturing solution) [Sambrook and Russel: Molecular Cloning. Volume 1, 7.4-7.8]:<\/li>\n<ul>\n<li>4M Guanidinium thiocyanate<\/li>\n<li>25mM Sodium citrate. 2H<sub>2<\/sub>O<\/li>\n<li>0.5% (w\/v) Sodium lauryl sarcosinate<\/li>\n<li>0.1M\u00a0\u03b2-mercaptoethanol.<\/li>\n<\/ul>\n<li>\u00a0For preparing 100 ml.<\/li>\n<ul>\n<li>Add 47.28g of Guanidinium thiocyanate in 50 mL H<sub>2<\/sub>O<\/li>\n<li>(Solution D is very caustic.\u00a0\u00a0 Wear appropriate gloves, a laboratory coat, and eye protection when preparing, handing, or working with this solution)<\/li>\n<li>5 ml of 10% sarcosyl<\/li>\n<li>3.3 ml of 0.75M sodium citrate pH 7<\/li>\n<li>Bring the volume to 100 ml with DEPC-treated H<sub>2<\/sub>O<\/li>\n<li>[<strong>Add\u00a0\u03b2-mercaptoethanol to an aliquot of solution D before each use<\/strong>]<\/li>\n<\/ul>\n<\/ol>\n<p>&nbsp;<\/p>\n<h2>RNA extraction protocol<\/h2>\n<ol start=\"1\">\n<li>Add liquid nitrogen to empty mortar and pestle, let it evaporate.<\/li>\n<li>Transfer all of the frozen tissue to a mortar containing liquid nitrogen and pulverize the tissue using a pestle.\u00a0 The tissue can be kept frozen during pulverization by addition of liquid nitrogen.<\/li>\n<li>Transfer the pulverized tissue to a polypropylene snap cap tube (falcon tubes), containing 3 ml of solution D. Add 22\u03bcl of\u00a0\u03b2-ME for 3ml of solution D.<\/li>\n<ul>\n<li>100 mg of tissue: 3 mL solution D. (Sambrook)<\/li>\n<li>100 mg of cells: 1mL\u00a0 solution D (Lab protocol).<\/li>\n<\/ul>\n<li>Sonicate 10 times for 1sec on high.\u00a0 This will vary with your sonciator. We use a microtip and set the sonicator on full power.\u00a0 Alternatively, material can be broken up by bead beating.<\/li>\n<li>Sequentially add:<\/li>\n<ul>\n<li>0.3 ml of 2M NaOAc pH 4. Mix.<br \/>\n[stock =3 M NaOAc.\u00a0 Use 0.2mL of 3M NaOAc pH 4- following the lab protocol]<\/li>\n<li>3 mL\u00a0 of phenol. Mix. (This should be around pH 6.0 &#8211; we use OmniPur Phenol (6705) without the addition of the supplied Tris:EDTA buffer)<\/li>\n<li>0.6 ml of chloroform: isoamyl alcohol (IAA) (49:1). (Prepare before you start extraction.)<\/li>\n<\/ul>\n<li>Mix well for 10 sec or longer.\u00a0 Solution should appear uniformly mixed.<\/li>\n<li>Chill on ice for 15 min.(Turn on centrifuge, change rotors and cool to 4\u00baC).<\/li>\n<li>Centrifuge at 10,000g for 20 min. at 4\u00baC. RNA is present in the aqueous phase.<\/li>\n<li>Transfer the upper aqueous phase to new tube and add equal volume of chloroform: IAA (49:1). Mix well and repeat centrifugation at 10,000g for 20 min at 4\u00baC. Repeat these steps until the interface is clear.<\/li>\n<ul>\n<li>[Note: To minimize contamination by DNA trapped at the interface, avoid taking the lowest part of the interface.]<\/li>\n<\/ul>\n<li>Transfer the aqueous phase to a clean tube. Add 3 mL of isopropanol. Place at -20\u00baC for 1 hr. [Depending on how much RNA precipitates you can decrease the time to \u00bd hr.]<\/li>\n<li>Centrifuge at 10,000 x g for 20 min at 4 \u00baC (30 min in Sambrook)<\/li>\n<li>Decant isopropanol. Air dry pellet. (Clean and dry RNA should be translucent).<\/li>\n<ul>\n<li>[Important: Pellets are easily lost. Decant the supernatant into a fresh tube. Do not discard it until the pellet has been checked.]<\/li>\n<\/ul>\n<li>Dissolve the pellet in 0.3 ml of solution D.<\/li>\n<li>Add equal volume of chloroform: IAA, mix thoroughly and centrifuge at 10,000g for 20 min. at 4\u00baC.<\/li>\n<li>Transfer the upper aqueous phase in a clean 1.5 ml tube and precipitate with 1 volume of isopropanol. Store at -20\u00baC for 1 hr and centrifuge at 10,000g for 20 min. at 4\u00baC.<\/li>\n<li>Decant the supernatant and wash with 70% ethanol. (~600\u03bcl) [This will get rid of the salts that came along during precipitation.]<\/li>\n<li>Centrifuge at 10,000g for 20 min. at 4\u00baC. Decant the supernatant and air dry the pellet.<\/li>\n<li>Dissolve the pellet it DEPC-H<sub>2<\/sub>O (use your own judgment depending on the pellet. ~50-100\u03bcl)\u00a0and store at -80\u00baC.<\/li>\n<\/ol>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>(Contributed by S. Ghoshroy, edited 11\/15\/2011 by DLR) Preparation of material: Read the following set of instructions before you plan to extract RNA.\u00a0 Solutions, and glassware should be ready before you take the samples out of the freezer for extraction.\u00a0 &hellip;<\/p>\n<p class=\"read-more\"> <a class=\"more-link\" href=\"https:\/\/wordpress.clarku.edu\/debrobertson\/laboratory-protocols\/rna-extraction-from-red-algae\/\"> <span class=\"screen-reader-text\">RNA Extraction from Red Algae<\/span> Read More &raquo;<\/a><\/p>\n","protected":false},"author":72,"featured_media":0,"parent":8,"menu_order":25,"comment_status":"closed","ping_status":"closed","template":"onecolumn-page.php","meta":{"ngg_post_thumbnail":0,"footnotes":""},"class_list":["post-228","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/228","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/users\/72"}],"replies":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/comments?post=228"}],"version-history":[{"count":0,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/228\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/8"}],"wp:attachment":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/media?parent=228"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}