{"id":231,"date":"2012-04-10T16:06:07","date_gmt":"2012-04-10T16:06:07","guid":{"rendered":"https:\/\/wordpress.clarku.edu\/debrobertson\/?page_id=231"},"modified":"2018-02-28T21:30:17","modified_gmt":"2018-02-28T21:30:17","slug":"reverse-transcription-rt-of-diatom-rna-using-qiagens-quantitect","status":"publish","type":"page","link":"https:\/\/wordpress.clarku.edu\/debrobertson\/laboratory-protocols\/reverse-transcription-rt-of-diatom-rna-using-qiagens-quantitect\/","title":{"rendered":"REVERSE TRANSCRIPTION (RT) OF DIATOM RNA USING QIAGEN’S QUANTITECT"},"content":{"rendered":"

Note<\/strong>: RNA should always be diluted in RNAse free water.<\/p>\n

    \n
  1. We generally use 1 \u00b5g of total RNA in the reverse transcription reaction.  For each sample, calculate the volume of  the purified RNA solution is required and how much water will be needed to bring the volume to 12 \u00b5L.  If the concentration of RNA is low, use 12 \u00b5L (and hope for the best). <\/li>\n
  2. Thaw RNA samples on ice. Make dilutions if necessary. Use RNase free water for dilutions.<\/li>\n
  3. Label PCR tubes and add 2 \u00b5L of gDNA wipeout buffer to each tube. Place the tubes on ice.<\/li>\n
  4. Add RNA or RNA+dH2<\/sub>O [total vol.=12\u03bcL] to the tubes containing the  gDNA wipeout buffer.<\/li>\n
  5. Incubate for 2 min at 42\u00baC in the PCR machine. Then place tubes immediately on ice.<\/li>\n
  6. Add 6\u03bcL of RT master mix to each of the RNA + gDNA mix. [Final vol. 20\u03bcL in each reaction.] Mix by pipetting up and down and store on ice.<\/li>\n
  7. In the PCR machine, incubate for 30 min at 42\u00baC then for 3 min at 95\u00baC to inactivate Quantiscript Reverse Transciptase.  Samples can be held at  4\u00ba C until removed from the PCR machine.<\/li>\n<\/ol>\n

    Store RT reaction on ice and proceed directly with Real-Time PCR, or for long term storage, store RT reactions at -20\u00baC.<\/p>\n","protected":false},"excerpt":{"rendered":"

    Note: RNA should always be diluted in RNAse free water. We generally use 1 \u00b5g of total RNA in the reverse transcription reaction.  For each sample, calculate the volume of  the purified RNA solution is required and how much water …<\/p>\n

    REVERSE TRANSCRIPTION (RT) OF DIATOM RNA USING QIAGEN’S QUANTITECT<\/span> Read More »<\/a><\/p>\n","protected":false},"author":72,"featured_media":0,"parent":8,"menu_order":30,"comment_status":"closed","ping_status":"closed","template":"","meta":{"ngg_post_thumbnail":0,"footnotes":""},"class_list":["post-231","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/231","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=231"}],"version-history":[{"count":0,"href":"https:\/\/wordpress.clarku.edu\/debrobertson\/wp-json\/wp\/v2\/pages\/231\/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=231"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}