Bitan:Autoradiography of hot gels

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TBE-Urea Gel Electrophoresis and Autoradiography of the Gels<o:p></o:p>

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1. Prepare the samples for electrophoresis using 1-μl aliquots of RNA before and after G-50 purification. <o:p></o:p>

2. Add 4 μl STE buffer and 5 μl 2× Novex® TBE-Urea Sample Buffer. <o:p></o:p>

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></o:p>

4. Assemble a 6% TBE-urea-polyacrylamide gel in the gel-running apparatus, fill the inside and outside chambers with 1× Novex® TBE-Urea Running Buffer or TBE-urea buffer made up in the lab.<o:p></o:p>

5. Rinse the wells of the precast gel using the buffer by a 1-ml pipettor.<o:p></o:p>

6. Centrifuge the RNA tubes and load the samples (10 µl total) using gel-loading tips. <o:p></o:p>

7. Run the gel at 180 V for 50 min.<o:p></o:p>

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></o:p>

9. Clean the surface of the work area with Decon making sure all the contaminating radioactive spots (if present) are removed. <o:p></o:p>

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></o:p>

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></o:p>

12. Leave the cassette at −20 °C for 60–90 min. <o:p></o:p>

13. Develop the film in the dark room under safe light after 60–90 min using an automatic film developer.<o:p></o:p>

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Bitan:Autoradiography of hot gels

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