Bitan:TBE-urea-polyacrylamide gel electrophoresis and autoradiography
TBE-urea-polyacrylamide gel electrophoresis of RNA product
1. Use the same 1-μl aliquots of RNA used for scintillation counting. Add 4 μl RNase-free water and 5 μl Novex® TBE-Urea Sample Buffer (2×). Heat the samples at 70 °C for 5 min (it is observed that this denaturing step is unnecessary for this analysis).
2. Assemble a 6% TBE-urea-polyacrylamide gel in the gel-running apparatus.
3. Wash the wells of the precast gel using the Novex® TBE-Urea Running Buffer. Or make up the TBE-urea buffer (see below).
4. Centrifuge the RNA samples; load the samples using gel-loading tips. Run the gel at 180 V for 50 min.
5. 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.
6. Clean the surface of the work area making sure to remove the contaminating radioactive spots on the work area.
7. Lay out two plies of plastic Saran wrap and wrap the gel and the plastic backing in the plastic wrap.
8. 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.
9. Leave the cassette at −20 °C for 60–90 min.
10. Develop the film in the dark room under safe light or in the dark after 60–90 min using the automatic Kodak film developer.
11. Scan the films and save the file.