If you have already made aliquots of dye, simply transfer your cDNA in bicarbonate buffer (10.5 mL) to the aliquot of dye. Alternatively, dissolve Cy dye in 10 ul DMSO and add 1 mL of dye to 10.5 mL of the cDNA reaction. 10% DMSO in the coupling reaction will not affect the chemical reaction. Aliquot unused dye and dry immediately.

Incubate 1 hour at RT in the dark. Mix every 15 minutes.

E. Quenching and Cleanup

Before combining Cy3 and Cy5 samples for hybridization, unreactive NHS-ester Cy dye must be quenched to prevent cross coupling.

Add 4.5 mL 4M hydroxylamine (Sigma).

Let reaction incubate 15 minutes in the dark.

To remove unincorporated/quenched Cy dyes, proceed with Qia-Quick PCR purification kit (QIAGEN). Method described below is as specified by manufacturer.

Combine Cy3 and Cy5 reactions.

Add 70 mL water.

Add 500 mL Buffer PB.

Apply to Qia-quick column and spin at 13K for 30-60 seconds. (optional: reapply flow-though for optimal binding).

Decant flow-through.

Add 750 mL Buffer PE and spin 30-60 seconds.