Protocol: DNA Probe Labeling by PCR

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.

* 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:

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:

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.