|
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.
|
