Bitan:TBE-urea-polyacrylamide gel electrophoresis
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<o:DocumentProperties> <o:Author>Farid Rahimi</o:Author> <o:Template>Normal</o:Template> <o:LastAuthor>Farid Rahimi</o:LastAuthor> <o:Revision>3</o:Revision> <o:Created>2009-12-03T18:04:00Z</o:Created> <o:LastSaved>2009-12-03T18:05:00Z</o:LastSaved> <o:Pages>1</o:Pages> <o:Company>UCLA</o:Company> <o:Lines>1</o:Lines> <o:Paragraphs>1</o:Paragraphs> <o:Version>11.1282</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings>
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mso-shading:windowtext;mso-pattern:solid #FFFF99;border-collapse:collapse; border:none;mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 5.4pt 0cm 5.4pt'> <tr> <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"'> </span></span><![endif]><span style='font-size:10.0pt;mso-fareast-language: KO'>Use the same 1-μl 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 μl RNase-free water and 5 μl <a href="http://products.invitrogen.com/ivgn/product/LC6876">Novex® TBE-Urea Sample Buffer (2×)</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"'> </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"'> </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"'> </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"'> </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"'> </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"'> </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"'> </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"'> </span><![endif]><span style='font-size:10.0pt;mso-fareast-language:KO'>Leave the cassette at −20 °C for 60–90 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"'> </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 60–90 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> </td> </tr>
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