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Star Republic: Guide for Biologists

Labeling PCR products with Biotin/Digoxigenin

1. Modified nucleotides: Biotin-16-dUTP or Digoxigenin-11-dUTP are used to label PCR products.

2. Make a nucleotide mix containing Biotin-16-dUTP or Digoxigenin-11-dUTP:

Biotin-16-dUTP or Digoxigenin-11-dUTP 350 mM
dTTP 650 mM
dATP 1 mM
dCTP 1 mM
dGTP 1 mM

3. Do a 50 ml PCR reaction, replacing dNTP with Biotin-16-dUTP or Digoxigenin-11-dUTP nucleotides mix:

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
Biotin-16-dUTP or Digoxigenin-11-dUTP nucleotides mix 5 ml
PCR DNA Polymerase (1 units/ml) 1 ml
DNA template (20-200 ng/ml) 1 ml
ddH2O 33 ml
Total volume 50 ml
4. See PCR for details of PCR reactions.