Bitan:Extraction of RNA from filters
<html xmlns:v="urn:schemas-microsoft-com:vml" xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns="http://www.w3.org/TR/REC-html40">
<head> <meta name=Title content="RNA extraction from the filters"> <meta name=Keywords content=""> <meta http-equiv=Content-Type content="text/html; charset=macintosh"> <meta name=ProgId content=Word.Document> <meta name=Generator content="Microsoft Word 11"> <meta name=Originator content="Microsoft Word 11"> <link rel=File-List href="RNA%20extraction%20from%20the%20filters_files/filelist.xml"> <title>RNA extraction from the filters</title> <!--[if gte mso 9]><xml>
<o:DocumentProperties> <o:Author>Farid Rahimi</o:Author> <o:Template>Normal</o:Template> <o:LastAuthor>Farid Rahimi</o:LastAuthor> <o:Revision>2</o:Revision> <o:Created>2009-12-18T23:36:00Z</o:Created> <o:LastSaved>2009-12-18T23:36:00Z</o:LastSaved> <o:Pages>1</o:Pages> <o:Words>403</o:Words> <o:Characters>2299</o:Characters> <o:Company>UCLA</o:Company> <o:Lines>19</o:Lines> <o:Paragraphs>4</o:Paragraphs> <o:CharactersWithSpaces>2823</o:CharactersWithSpaces> <o:Version>11.1282</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings>
</xml><![endif]--><!--[if gte mso 9]><xml>
<w:WordDocument> <w:DisplayHorizontalDrawingGridEvery>0</w:DisplayHorizontalDrawingGridEvery> <w:DisplayVerticalDrawingGridEvery>0</w:DisplayVerticalDrawingGridEvery> <w:UseMarginsForDrawingGridOrigin/> </w:WordDocument>
</xml><![endif]--> <style> <!--
/* Font Definitions */
@font-face {font-family:"Times New Roman"; panose-1:0 2 2 6 3 5 4 5 2 3; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} @font-face {font-family:"Lucida Grande"; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} @font-face {font-family:Calibri; panose-1:0 2 15 5 2 2 2 4 3 2; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} @font-face {font-family:"Arno Pro Italic"; panose-1:0 2 2 5 2 4 5 6 9 4; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;}
/* Style Definitions */
p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin-top:0cm; margin-right:0cm; margin-bottom:10.0pt; margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri;} a:link, span.MsoHyperlink {color:blue; text-decoration:underline; text-underline:single;} a:visited, span.MsoHyperlinkFollowed {color:purple; text-decoration:underline; text-underline:single;} table.MsoNormalTable {mso-style-parent:""; font-size:10.0pt; font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> </style> <!--[if gte mso 9]><xml>
<o:shapedefaults v:ext="edit" spidmax="1026"/>
</xml><![endif]--><!--[if gte mso 9]><xml>
<o:shapelayout v:ext="edit"> <o:idmap v:ext="edit" data="1"/> </o:shapelayout></xml><![endif]-->
</head>
<body bgcolor=white lang=EN-US link=blue vlink=purple style='tab-interval:36.0pt'>
<div class=Section1>
<p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height: normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'><b>RNA extraction from the filters<o:p></o:p></b></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>RNA is extracted from the filters to obtain the sequences that bind to the protein. These sequences are amplified for the next SELEX cycle.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>1) After <a href="http://openwetware.org/wiki/Bitan:Scintillation_counting_using_the_Triathler_bench-top_counter">scintillation counting</a>, remove the positive-control filter from the Eppendorf tube (<a href="http://openwetware.org/wiki/Bitan:Filter_binding_for_SELEX">from previous experiment</a>) and place into a clean, dry, </span><span style='font-size: 14.0pt;font-family:"Arno Pro Italic";mso-fareast-language:KO'>35×10-mm </span><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>Petri dish. <o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>2) Use a clean scalpel and a pair of tweezers to cut the membrane in small pieces.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>3) Using the tweezers, transfer the cut pieces of the membrane into the same labeled Eppendorf tube from Step 1.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>4) Add 400 μl elution buffer (7 M urea, 3 mM ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), 100 mM sodium citrate, pH 5.0) and incubate the tube at 95 °C for 10 min.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>5) Centrifuge the tube at top speed, aspirate, and collect the extraction butter into a new labeled Eppendorf tube.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>6) Measure the remaining radioactivity counts in the tube containing the membrane pieces by scintillation counting to assess the extraction efficiency.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>7) Repeat the extraction process (steps 4–6) thrice. The efficiency after 3 extractions is usually ~95–96%.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>8) In the tubes containing the RNA e</span><span style='font-size:14.0pt;font-family:"Arno Pro Italic"; mso-fareast-language:KO'>xtracts, add 1 volume (400 μl) of citrate-saturated phenol (pH 4.7):chloroform:isoamyl alcohol (125:24:1). Mix by a vortex for ~1 minute and centrifuge at 16,000 <i>g</i></span><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'> for 2 minutes.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'>9) Transfer the upper, aqueous phase to a fresh tube or discard the bottom phase by aspiration using a micropipette. <o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'>10) Add 1 volume of chloroform:isoamyl alcohol (24:1), mix by a vortex for 1 minute and centrifuge at 16,000 <i>g</i></span><span style='font-size:14.0pt; font-family:"Arno Pro Italic";mso-fareast-language:KO'> for 2 min. <o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'>11) Transfer the upper, aqueous phase to a fresh tube or discard the bottom phase by aspiration using a micropipette. <o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'>12) To precipitate the RNA, add 0.1 volume of 3 M sodium acetate (pH 5.2), 3–4 μl glycogen (10 μg/μl) as the coprecipitant, and 1 volume equivalent of propan-2-ol. Mix and place in a −20 °C freezer overnight. <o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'>13) Spin at top speed, preferably in a microcentrifuge at 4 °C, for 20–30 minutes to precipitate the RNA from step 12.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'>14) After centrifugation, aspirate the supernate carefully without dislodging the coprecipitant phase barely visible in the tube. <o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic";mso-fareast-language: KO'>15) Wash the RNA pellet with 0.5 ml of 70% ethanol, centrifuge for 5 min a top speed and discard ethanol by aspiration without dislodging the coprecipitant phase barely visible in the tube.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'>16) Dissolve the RNA pellet in 50 μl STE buffer and proceed to <a href="http://openwetware.org/wiki/Bitan:In-vitro_transcription%2C_labeling_and_G-50_purification_of_RNA">G-50 purification</a>.<o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm; margin-left:36.0pt;margin-bottom:.0001pt;line-height:normal'><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><a href="http://openwetware.org/wiki/Bitan:todo">Back to To-Do List</a>.<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-size:14.0pt;font-family:"Arno Pro Italic"'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p>
</div>
</body>
</html>