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.
- 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®.
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:
|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.
- Fluorescent dyes may cause shifts in DNA migration of the labeled DNA compared to unlabeled PCR product.