Bitan:TBE-urea-polyacrylamide gel electrophoresis and autoradiography



       TBE-urea-polyacrylamide gel electrophoresis of RNA product



   TBE-urea-polyacrylamide gel electrophoresis of RNA product <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. <![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>

<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. <![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> <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. <![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>

<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. <![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> <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. <![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> <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. <![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>

<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. <![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> <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. <![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> <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"'>    <![endif]><span style='font-size:10.0pt;mso-fareast-language:KO'>Leave the cassette at &#8722;20 °C for 60&#8211;90 min. </o: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"'> <![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. </o: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]>11.<span style='font:7.0pt "Times New Roman"'> <![endif]><span style='font-size:10.0pt;mso-fareast-language:KO'>Scan the films and save the file. <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>

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