Biotin-16-dUTP Protocol & Troubleshooting

Product No. 11093070910

Protocol

PCR-labeling with Biotin-16-dUTP

The incorporation of Biotin-16-dUTP using PCR is possible with most DNA polymerases, e.g., as described for DIG-dUTP in the Package Insert of Taq DNA Polymerase.

More information is available in the Roche PCR Manual and the Roche DIG Application Manual for Filter Hybridization.

Troubleshooting

Evaluation of Labeling Efficiency

There is no biotin-labeled control DNA available. For a rough estimation of labeling efficiency, use a dilution series in a direct spot test, similar to the method used to evaluate DIG-labeled probes.

After estimating the labeling concentration set up a dilution series of the labeled DNA probe. For blocking of the membrane, use 5% Blocking Reagent.

Instead of the anti-DIG antibody Streptavidin-AP (alkaline phosphatase) conjugate, Prod. No. 11093266910, use a 1:5000 dilution for direct colorimetric or luminescent detection of the biotin-labeled DNA probe spotted on the membrane.