Endy:Northern blot, AlkPhos end-labeled probes

The following protocol for Northern Blotting is based on the RNA extraction method used by Sean Moore, Agarose gel electrophoresis, and the Turboblotter system from Whatman.

Materials

• RNA extracted from cells (should be ~1-5µg/µl).
• RNAse free water (make lots for rinsing glassware and electrophoresis chambers)
  • Add DEPC to final concentration of 0.1%.
  • Incubate 1hr at 37°C.
  • Autoclave for 15 mins at 15 psi.
• 10X BPTE electrophoresis buffer (The final pH of this 10x buffer is ~6.5.)
  • 100 mM PIPES
  • 300 mM Bis-Tris
  • 10 mM EDTA
• HPLC grade or better DMSO
• Glyoxal
  • Commercially available stock solutions of glyoxal contain both hydrated forms of glyoxal and oxidation products that can degrade RNA. These must be removed.
• Glyoxal reaction mixture (divide into small aliquots and store at -70°C)
  • 6mL DMSO
  • 2mL deionized glyoxal
  • 1.2mL of 10X BPTE electrophoresis buffer
  • 0.6mL of 80% glycerol
• RNA size marker
• RNA gel loading buffer
  • 95% deionized formamide
    • Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
  • 0.025% (w/v) bromophenol blue
  • 0.025% (w/v) xylene cyanol FF
  • 5 mM EDTA (pH 8.0)
  • 0.025% (w/v) SDS

• SYBR Gold