Bitan:Autoradiography of hot gels
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TBE-Urea Gel
Electrophoresis and Autoradiography of the Gels
1. Prepare the samples for
electrophoresis using 1-μl aliquots of RNA before and after G-50
purification.
2. Add 4 μl STE buffer and 5
μl 2× Novex® TBE-Urea Sample Buffer.
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).
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.
5. Rinse the wells of the precast gel
using the buffer by a 1-ml pipettor.
6. Centrifuge the RNA tubes and load the
samples (10 µl total) using gel-loading tips.
7. Run the gel at 180 V for 50 min.
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.
9. Clean the surface of the work area
with Decon making sure all the contaminating radioactive spots (if present) are
removed.
10. Lay out two layers of plastic Saran
wrap. Wrap the gel and the plastic backing in between the two-plied plastic
wrap.
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.
12. Leave the cassette at −20 °C
for 60–90 min.
13. Develop the film in the dark room
under safe light after 60–90 min using an automatic film developer.
