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