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Star Republic: Guide for Biologists

Labeling of DNA --- Amino-allyl dye coupling

PCR

1. For a 50 ml PCR reaction:

10X PCR Reaction Buffer 5 ml
Forward primer (20 mM, 20 pmol/ml ) 1 ml
Reverse primer (20 mM, 20 pmol/ml ) 1 ml
25 mM MgCl2 3 ml
10 mM dNTP (mix of 10 mM dATP, dCTP, dGTP each) 1 ml
4 mM dTTP 1 ml
6 mM aminoallyl-dUTP 1 ml
PCR DNA Polymerase (1 units/ml) 1 ml
DNA template (1-10 ng/ml) 1 ml
ddH2O 35 ml
Total volume 50 ml

2. Dilute and purifying the labeled cDNA using Microcon-30 filter.

Warning: buffers containing free amine groups (e.g., Tris) interfer with the coupling reactions.

3. Concentrate to 10 ml.

4. Coupling to monofunctional Cy3/Cy5 dye as described in Labeling of cDNA --- Amino-allyl dye coupling.