Bitan:TBE-urea-polyacrylamide gel electrophoresis

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<head> <meta name=Title content="TBE-urea-polyacrylamide gel electrophoresis of RNA product"> <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="TBE-Urea%20Gel_files/filelist.xml"> <title>TBE-urea-polyacrylamide gel electrophoresis of RNA product</title> <!--[if gte mso 9]><xml>

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<table border=1 cellspacing=0 cellpadding=0 style='background:#FFFF99;

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 <td width=443 valign=top style='width:442.8pt;border:solid windowtext .5pt;
 padding:0cm 5.4pt 0cm 5.4pt'>
 <p class=MsoNormal align=center style='margin-top:0cm;margin-right:0cm;
 margin-bottom:0cm;margin-left:36.0pt;margin-bottom:.0001pt;text-align:center;
 line-height:normal'><span style='mso-fareast-language:KO'><b>TBE-urea-polyacrylamide
 gel electrophoresis of RNA product<o:p></o:p></b></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language:
 KO'>Use the same 1-&#956;l aliquots of RNA used for <a
 href="http://openwetware.org/wiki/Bitan:Scintillation_counting_using_the_Triathler_bench-top_counter">scintillation
 counting</a>. Add 4 &#956;l RNase-free water and 5 &#956;l <a
 href="http://products.invitrogen.com/ivgn/product/LC6876">Novex® TBE-Urea
 Sample Buffer (2&times;)</a>. Heat the samples at 70 °C for 5 min (it is
 observed that this denaturing step is unnecessary for this analysis). <o:p></o:p></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language:
 KO'>Assemble a 6% TBE-urea-polyacrylamide gel in the <a
 href="http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Protein-Gel-Electrophoresis/1D-Electrophoresis/Xcell-SureLock-Mini-Cell.html">gel-running
 apparatus</a>. <o:p></o:p></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language:
 KO'>Wash the wells of the precast gel using the <a
 href="http://products.invitrogen.com/ivgn/product/LC6675">Novex® TBE-Urea
 Running Buffer.</a> Or make up the TBE-urea buffer (see below).<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>4.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language:
 KO'>Centrifuge the RNA samples; load the samples using gel-loading tips. Run
 the gel at 180 V for 50 min.<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>5.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language:
 KO'>After 50 min, disassemble the gel by breaking apart the plastic cover of
 the precast gel and remove the shorter side leaving the longer backing as a
 support for the gel.<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>6.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language:
 KO'>Clean the surface of the work area making sure to remove the
 contaminating radioactive spots on the work area. <o:p></o:p></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>7.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language:
 KO'>Lay out two plies of plastic Saran wrap and wrap the gel and the plastic
 backing in the plastic wrap.<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>8.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;
 </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language:
 KO'>Expose the gel wrapped in plastic to an autoradiography X-ray film in the
 dark room under safe light (for Amersham films) or in the dark (for Denville
 films) using an exposure cassette.<o:p></o:p></span></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]>9.<span
 style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp; </span><![endif]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>Leave the cassette at
 &#8722;20 °C for 60&#8211;90 min.</span><o:p></o:p></p>
 <p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;
 margin-left:54.0pt;margin-bottom:.0001pt;text-indent:-18.0pt;line-height:
 normal;mso-list:l1 level1 lfo3;tab-stops:list 54.0pt'><![if !supportLists]>10.<span
 style='font:7.0pt "Times New Roman"'>&nbsp; </span><![endif]><span
 style='font-size:10.0pt;mso-fareast-language:KO'>Develop the film in the dark
 room under safe light or in the dark after 60&#8211;90 min using the
 automatic Kodak film developer.</span></p>
 <p class=MsoNormal align=center style='text-align:center'><a
 href="http://openwetware.org/wiki/Bitan:todo">Back to To-Do List</a><o:p></o:p></p>
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