Bitan:Autoradiography of hot gels



       TBE-Urea Gel Electrophoresis and Autoradiography of the Gels



 TBE-Urea Gel Electrophoresis and Autoradiography of the Gels

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<span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>1. Prepare the samples for electrophoresis using 1-&#956;l aliquots of RNA before and after G-50 purification. </o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>2. Add 4 &#956;l STE buffer and 5 &#956;l 2&times; Novex® TBE-Urea Sample Buffer. </o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>3. Heat the samples at 70 °C for 5 min (heating was found to be unnecessary because the gel resolution of the RNA samples with and without heating is the same in this experimental setup).</o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>4. Assemble a 6% TBE-urea-polyacrylamide gel in the gel-running apparatus, fill the inside and outside chambers with 1&times; Novex® TBE-Urea Running Buffer or TBE-urea buffer made up in the lab.</o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>5. Rinse the wells of the precast gel using the buffer by a 1-ml pipettor.</o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>6. Centrifuge the RNA tubes and load the samples (10 µl total) using gel-loading tips. </o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>7. Run the gel at 180 V for 50 min.</o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>8. After 50 min, disassemble the gel; break apart the plastic mold, remove and discard only the shorter backing of the gel mold, but leave the longer backing as a support for the gel.</o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>9. Clean the surface of the work area with Decon making sure all the contaminating radioactive spots (if present) are removed. </o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>10. Lay out two layers of plastic Saran wrap. Wrap the gel and the plastic backing in between the two-plied plastic wrap.</o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>11. Expose the gel wrapped in plastic to a sheet of autoradiography X-ray film inside an exposure cassette in the dark room, under safe light. </o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>12. Leave the cassette at &#8722;20 °C for 60&#8211;90 min. </o:p>

<p class=MsoNormal style='margin-left:36.0pt'><span style='font-size:14.0pt; color:#993366;mso-fareast-language:KO'>13. Develop the film in the dark room under safe light after 60&#8211;90 min using an automatic film developer.</o:p>

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