Posthybridization Washes

TNE (0.5 M NaCl, 10 mM Tris-HCl, 5 mM EDTA, pH 8.0). High-stringency buffer: 50% v/v formamide, 2X SSC, 10 mM DTT prepared fresh on day of use using solid DTT or a 100-mM stock of DTT stored at -20°C. RNase A (10 mg/mL) in water. Store at -20°C.

Graded (30, 50, 70, and 95% v/v) ethanols containing 0.3 M (final) ammonium acetate. The presence of the ammonium acetate ensures that probe-target RNA duplexes remain intact during the dehydration process. Duplexes are stable in 100% (v/v) ethanol.

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