biotechrabbit™ Heat Labile Uracil–DNA Glycosylase selectively degrades uracil-containing PCR products. After performing PCR or RT-PCR using dUTP instead of dTTP, PCR products remain intact after treatment with Heat Labile Uracil–DNA Glycosylase, whereas contaminating DNA (i.e., not amplified) is degraded. Heat Labile Uracil–DNA Glycosylase is completely and irreversibly inactivated by moderate heat treatment at 50⁰C, allowing contamination control in RT-qPCR. The enzyme hydrolyses the N-glycosylic bond between the deoxyribose sugar and the base in uracil-containing DNA leaving an abasic (apyrimidinic) site in DNA but does not modify uracils in RNA.

Heat Labile Uracil–DNA Glycosylase is highly active at 20–40°C. No cofactors or divalent cations are required for activity, and the enzyme is active in most PCR and RT-PCR buffers. Although the enzyme is active a pH 6.5–9.0, the optimal pH 7.5 is in 50 mM NaCl.

Unit Definition

One Unit will liberate 1 nmol Uracil from Uracil-containing DNA per hour at 37°C.

Quality Control

Protein Purity

Purified to apparent homogeneity by SDS-PAGE. No nuclease activity is detected.

Heat Labile Uracil–DNA Glycosylase is active in all commercially available PCR master mixes. Previous PCR/RT-PCR reactions must have used dUTP containing dNTP mixes.

PCR/QPCR

• Add 0.25 µl (0.25 U) Heat Labile Uracil–DNA Glycosylase to a 25 µl PCR reaction
• Incubate for 5 min at room temperature
• Start the PCR run

One-step RT-PCR

• Add 0.2 µl (0.2 U) Heat Labile Uracil–DNA Glycosylase to a 20 µl RT-PCR reaction
• Incubate for 5 min at room temperature
• Run the reverse transcription at 50- 55°C
• Start the PCR run

Store final PCR/RT-PCR products at -20°C or 4°C degrees.

Product manual

Other documentation