Labelling PCR Products with Digoxigenin

PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concentration in a nucleotide mix of final concentration 50-100uM dNTPs (Emanual, 1991; Nucleic Acids Res 19: 2790). This allows substitution to a known extent of probes of exactly defined length, which in turn allows exactly defined bybridisation conditions. It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling.

Make a DIG-dNTP mix for PCR as follows:

DIG NUCLEOTIDE MIX CONCENTRATIONS

• Dig-11-dUTP 350 uM
• dTTP 650 uM
• dATP 1 mM
• dCTP 1 mM
• dGTP 1 mM

For each 50 ul of probe synthesized, a 1/10 dilution is made of the DIG-nucleotide mix when added to the other reagents as described above. The products may be analyzed by agarose gel electrophoresis - NOTE: PRODUCTS ARE LARGER THAN NON-SUBSTITUTED PRODUCT - and detected directly on blots immunologically. Probes can be used as 5-10 ul aliquots directly from PCR product mixes, mixed with hybridisation mix and denatured. Probes can be re-used up to 10 times, stored frozen in between experiments and boiled to denature.