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Star Republic: Guide for Biologists

Labeling of cDNA --- Amino-allyl dye coupling

RT

1. Mix 1-2 mg total RNA in nuclease-free ddH2 with 2 mg Oligo-dT. Add control RNA if necessary. Adjust total volume to 13 ml with nuclease-free ddH2.

If using mRNA, replace Oligo-dT with random primers.

Concentrating the RNA sample if necessary.

2. Heat to 70 C for 5-10 minutes. Cool on ice for 5 minutes.

3. Add 12 ml of the following mixture:

5X RT Buffer 5 ml
100 mM DTT 2.5 ml
RNase Inhibitor 0.5 ml
dATP, dCTP, dGTP (25 mM each) 0.5 ml
5 mM dTTP 0.5 ml
5 mM aminoallyl-dUTP 2 ml
Superscript RT II 1 ml

4. Mix and incubate at 42 C for 1-4 hours.

Hydrolysis and purification

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

6. Neutralize by addition of 15 ml 0.1 N HCl.

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

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

8. Concentrate to 10 ml.

Coupling

9. Add 0.5 ml 1M sodium bicarbonate (pH 9.0).

10. Add 2 ml NHS-ester Cy3 or Cy5 dye.

11. Incubate at room temperature for 1 hour in the dark.

12. Quenching by adding 4.5 ml 4 M hydroxylamine.

13. Incubate at room temperature for 15 minutes in the dark.

14. Dilute and purifying the labeled cDNA using QIAquick or Microcon-30 filter.

Coupling efficiency

15. OD measurements may be used to estimate the amount of cDNA and incorporated Cy3/Cy5. You may need a cuvette with 10 ml volume.

  • cDNA amount = A260 * 37 mg/ml * volume
  • Cy3 molecules/1000 bases = 58.5 * A550 / A260
  • Cy5 molecules/1000 bases = 35.1 * A650 / A260

    Or

  • Cy3 incorporated (pmols) = A550 * volume (ml) / 0.15
  • Cy3 incorporated (pmols) = A650 * volume (ml) / 0.25