Bitan:TBE-urea-polyacrylamide gel electrophoresis and autoradiography

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TBE-urea-polyacrylamide gel electrophoresis of RNA product<o:p></o:p>

<![if !supportLists]>1.     <![endif]>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>

<![if !supportLists]>2.     <![endif]>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>

<![if !supportLists]>3.     <![endif]>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>

<![if !supportLists]>4.     <![endif]>Centrifuge the RNA samples; load the samples using gel-loading tips. Run the gel at 180 V for 50 min.<o:p></o:p>

<![if !supportLists]>5.     <![endif]>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>

<![if !supportLists]>6.     <![endif]>Clean the surface of the work area making sure to remove the contaminating radioactive spots on the work area. <o:p></o:p>

<![if !supportLists]>7.     <![endif]>Lay out two plies of plastic Saran wrap and wrap the gel and the plastic backing in the plastic wrap.<o:p></o:p>

<![if !supportLists]>8.     <![endif]>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>

<![if !supportLists]>9.     <![endif]>Leave the cassette at −20 °C for 60–90 min.<o:p></o:p>

<![if !supportLists]>10.  <![endif]>Develop the film in the dark room under safe light or in the dark after 60–90 min using the automatic Kodak film developer.<o:p></o:p>

<![if !supportLists]>11.  <![endif]>Scan the films and save the file.

<a href="http://openwetware.org/wiki/Bitan:todo">Back to To-Do List</a><o:p></o:p>

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