This protocol can be adapted to use dUTP or dCTP labeled with other dyes, biotin, or haptens like digoxigenin. See our full selection of labeled nucleotides.
Materials required:
- Molecular biology grade water
- Taq DNA polymerase *
- 10X Taq reaction buffer
- 25 mM MgCl2
- dATP, dTTP, dCTP, dGTP (separate stock solutions)
- CF® Dye dUTP or CF® Dye dCTP
- DNA template
- Forward and reverse primers, 10 uM each
- PCR clean-up kit or G50 Sephadex® microspin column (optional)
Workflow overview:
- Set up labeling reactions
- Perform PCR amplification
- Remove unincorporated nucleotides (optional)
- Evaluate labeling by gel electrophoresis
* When using dUTP conjugates for labeling, use Taq DNA polymerase; dUTP inhibits archaeal polymerases such as Pfu and Vent®.
Procedure:
1 – Set up labeling reactions
1.1 For each labeling reaction, set up the PCR reaction mix as shown below:
| Component | Volume per reaction | Final concentration* |
|---|---|---|
| 10X Taq reaction buffer | 2 uL | 1X |
| 25 mM MgCl2 | 2 uL | 5 mM |
| 1 mM dATP | 2 uL | 100 uM |
| 1 mM dCTP | 2 uL | 100 uM |
| 1 mM dGTP | 2 uL | 100 uM |
| 1 mM dTTP | 1 uL | 50 uM |
| 10 uM forward primer | 1 uL | 500 nM |
| 10 uM reverse primer | 1 uL | 500 nM |
| Template DNA | 1 ng | 50 pg/uL |
| Taq | 1 U | 0.05 U/uL |
| Molecular grade dH20 | to 19 uL total |
1.2 Add 1 uL of 1 mM CF® dye dUTP to the reaction tube.
- Optional: for an unlabeled control reaction, add 1 uL of 1 mM dTTP instead of CF® dye dUTP.
- If using fluorescent dCTP, set up the reaction with 50 uM dCTP and 100 uM dTTP, then add 1 uL of 1 mM CF® Dye dCTP to the reaction.
2 – Perform PCR amplification
Amplify the reactions in a thermocycler using the following cycling protocol:
| Step | # Cycles |
|---|---|
| Denaturing/Taq activation 94°C, 2 min.1 | Hold |
| Denaturing 94°C 30 sec. | Cycle 30X |
| Annealing 30 sec.2 | |
| Extension 72°C 1 min. 3 | |
| Final extension 72°C 5 min. | Hold |
2. Set the annealing temperature 5°C below the melting temperature (Tm) of your primers.
3. This cycling protocol was optimized for 200-300 bp amplicons. Longer amplicons may require longer extension times.
3 -Remove unincorporated nucleotides
Use a PCR clean-up kit or G50 Sephadex® microspin column to remove unincorporated nucleotides.
- Removal of unincorporated nucleotides may not be necessary before hybridization, but the fluorescence from free labeled nucleotides can make it difficult to evaluate labeled PCR products by gel electrophoresis.
4 – Evaluate labeling by gel electrophoresis
4-1 Run 10% of the labeled product on an agarose gel along with a DNA ladder. Do not add fluorescent DNA dye to the agarose before casting. After electrophoresis, image the CF® dye fluorescence of the labeled probes on a UV gel transilluminator or laser-based gel scanner as appropriate for the wavelengths of the specific dye used.
- It can be useful to run an unstained DNA ladder in one lane, and a DNA ladder prestained with GelRed® Prestain Plus 6X DNA Loading Buffer in another lane to visualize the ladder before staining the entire gel.
- Visible fluorescent dyes (CF®405S to CF®594) can be viewed with UV excitation. Far-red fluorescence emission (650 nm or longer) is not visible to the human eye, but can be imaged using a fluorescence gel scanner using the appropriate excitation and emission settings.
- Be sure to image CF® dye fluorescence before staining DNA with gel stain, because CF® dye fluorescence may overlap with gel stain fluorescence, or CF® dyes and gel stains may quench one another.
4-2 After imaging the probe fluorescence, post-stain the gel with a nucleic acid gel stain like GelRed® or GelGreen® to image unstained DNA ladder and unlabeled control PCR product.
- Fluorescent dyes may cause shifts in DNA migration of the labeled DNA compared to unlabeled PCR product.