{"id":219,"date":"2012-04-10T15:53:16","date_gmt":"2012-04-10T15:53:16","guid":{"rendered":"https:\/\/wordpress.clarku.edu\/debrobertson\/?page_id=219"},"modified":"2012-05-23T17:18:30","modified_gmt":"2012-05-23T17:18:30","slug":"rna-extraction-from-diatoms-using-the-rneasy-kit","status":"publish","type":"page","link":"https:\/\/wordpress.clarku.edu\/debrobertson\/laboratory-protocols\/rna-extraction-from-diatoms-using-the-rneasy-kit\/","title":{"rendered":"RNA EXTRACTION FROM DIATOMS USING THE RNEASY KIT"},"content":{"rendered":"

A. RNA Extraction Protocol<\/h2>\n
    \n
  1. Determine how many samples will be analyzed.\u00a0\u00a0Each sample will require 600 \u00b5L of Buffer RLT and Buffer RLT requires the fresh addition of\u00a0b\u00a0– mercaptoethanol (add 10 \u00b5L of\u00a0b\u00a0– mercaptoethanol per 1 mL of Buffer RLT).\u00a0\u00a0This solution is stable for approximately 1 month.<\/li>\n
  2. For 5 x 107<\/sup>\u00a0\u2013 5 x 109<\/sup>\u00a0cells, resuspend pellet in 600 \u00b5L of Buffer RLT.<\/li>\n
  3. Sonicate for 10 x 1 sec pulses<\/li>\n
  4. Centrifuge for 3 min at maximum speed in a microcentrifuge and transfer the supernatant to a new, labeled tube.<\/li>\n
  5. Add and equal volume of 70% ethanol to the cleared lysate and mix well by pipetting.<\/li>\n
  6. Apply 700 \u00b5L of sample to a labeled RNeasy mini spin column sitting in a 2 mL collecting tube. Centrifuge for 15 sec at 8,000 x\u00a0g<\/em>\u00a0(> 10,000 rpm)<\/li>\n
  7. Repeat as needed until the entire sample has been loaded onto the column.<\/li>\n
  8. Pipet 700 \u00b5L of Buffer RW1 onto the column, centrifuge as above.<\/li>\n
  9. Discard flow through and collection tube<\/li>\n
  10. Transfer RNeasy column to new collection tube.\u00a0\u00a0Pipet 500 \u00b5L of Buffer RPE onto the column and centrifuge as above (ensure that ethanol has been added to Buffer RPE before use). Discard flow through<\/li>\n
  11. Pipet 500 \u00b5L of Buffer RPE onto RNeasy column and centrifuge for 2 min at maximum speed to dry the RNeasy membrane.\u00a0\u00a0Discard flow through and repeat<\/li>\n
  12. Transfer RNeasy to new 1.5 mL RNase-free microcentrifuge tube.\u00a0\u00a0Pipet 30-50 \u00b5L of RNase-free water directly onto the RNeasy membrane.\u00a0\u00a0Centrifuge for 1 min at >8,000 x g to elute RNA<\/li>\n
  13. Store eluted material at -80\u00b0C<\/li>\n<\/ol>\n

     <\/p>\n

    B. Working with RNA \u2013 Some precautions<\/h2>\n

    RNA is relatively easy to isolate from prokaryotic and eukaryotic cells.\u00a0\u00a0However, RNA molecules are highly susceptible to degradation by RNases, which are produced by the cell and on your skin.\u00a0\u00a0The general strategy for successful isolation of intact, full length mRNA is to minimize the possibility of RNase contamination.\u00a0\u00a0The following recommendations will reduce the potential of contamination:<\/p>\n

      \n
    1. Wear gloves at all stages of the extraction and when handling equipment used in the manipulation of RNA<\/li>\n
    2. Bake all glassware at 275 C for 3-4 h.\u00a0\u00a0This serves to burn of all organic material (including RNases) from the glass surface.\u00a0\u00a0Sterile, disposable polypropylene tubes and pipets are also useful.<\/li>\n
    3. Treat solutions (including water) with 0.05% diethyl pyrocarbonate (DEPC).\u00a0\u00a0DEPC is an effective protein denaturant and inactivates any residual RNases.\u00a0\u00a0Compounds containing amine groups (e.g. TRIS) cannot be DEPC treated and must be made from ultrapure stocks and DEPC-treated water.<\/li>\n
    4. Keep fingers and dirty (unbaked) utensils out of RNase free chemical stocks.\u00a0\u00a0I recommend pouring out dry chemicals into an RNase free container rather than \u201cscooping\u201d them out of the container.<\/li>\n
    5. Make sure your work surfaces are clean and wiped down with ethanol.<\/li>\n<\/ol>\n

      If these precautions are kept in mind, the success of your RNA isolation will be increased 100-fold.<\/p>\n","protected":false},"excerpt":{"rendered":"

      A. RNA Extraction Protocol Determine how many samples will be analyzed.\u00a0\u00a0Each sample will require 600 \u00b5L of Buffer RLT and Buffer RLT requires the fresh addition of\u00a0b\u00a0– mercaptoethanol (add 10 \u00b5L of\u00a0b\u00a0– mercaptoethanol per 1 mL of Buffer RLT).\u00a0\u00a0This solution …<\/p>\n

      RNA EXTRACTION FROM DIATOMS USING THE RNEASY KIT<\/span> Read More »<\/a><\/p>\n","protected":false},"author":72,"featured_media":0,"parent":8,"menu_order":15,"comment_status":"closed","ping_status":"closed","template":"onecolumn-page.php","meta":{"ngg_post_thumbnail":0,"footnotes":""},"class_list":["post-219","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/219","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=219"}],"version-history":[{"count":0,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/219\/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=219"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}