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Gel Purification of RNA

Elution Buffer Stock Final
10mM Tris-HCl pH 8 1M 6 μl
1mM EDTA 0.5M 1.2 μl
0.75M NH4OAc 10M 45 μl
0.1% SDS 20% 3 μl
DEPC H20 544.8 μl
  1. Pour a 6% acrylamide/7M urea gel (add 550 μl 10% APS and 55 μl Temed to 55ml of mix).
  2. Prerun gel at 40W, 30 min.
  3. Heat RNA to 90°C, 5 min in an equal volume of denaturing buffer (90% formamide, 1X TBE, BPB, XC).
  4. Remove urea from wells, load samples.
  5. Run at 40W.
  6. For radioactive RNA, wrap in saran wrap and expose to film 3-30 min (mark film with high energy pen).
  7. For nonradioactive samples use UV light to detect RNA.
  8. Excise the RNA with a clean razor blade. Use RNase Zap treated forceps to place in eppie.
  9. Add 500-600ul of Elution buffer.
  10. Rock overnight at 4°C.
  11. Transfer liquid to a fresh eppie, add NH4OAc to 2M (~80μl) and 1 volume of cold isopropanol. Precipitate at -20°C at least an hour.
  12. Spin 30-45 min and remove super. Wash pellet with 500μl 70% Ethanol. Spin 5 min. Dry briefly and resuspend pellet in 25μl of DEPC water or TE. Store hot RNA at -20°C and cold RNA at -80°C.

Download Protocol: Gel Purification of RNA
(note requires Microsoft Word to open)