B. Hydrolysis

1. Degrade RNA by addition of 15 mL of 0.1 N NaOH. Incubate at 70C for 10 minutes

2. Neutralize by addition of 15 mL 0.1 N HCl.

To continue with the amino-allyl dye coupling procedure, all Tris must be removed from the reaction to prevent the monofunctional NHS-ester Cy-dyes from coupling to free amine groups in solution.

3. Add 450 mL water to each reaction.

C. Cleanup

Add 500 mL neutralized, diluted reaction mix to a Microcon-30 filter (Amicon).

Spin at 12g for 7 minutes.

Discard flow through.

Repeat process two more times, refilling original filter with 450 mL water. Concentrate to 10 mL. Samples can be stored at -20C indefinitely.

D. Coupling

Add 0.5 mL 1M sodium bicarbonate, pH 9.0 to 50 mM final. Check 1M stock solution periodically for fluctuations in pH.

Monofunctional NHS-ester Cy3 (PA23001) and Cy5 dye (PA25001, Amersham) is supplied as a dry pellet. Each tube is sufficient to label 10 reactions under normal conditions.