Cod Uracil-DNA Glycosylase

The main advantages of Cod UNG

• Heat-labile, completely and irreversibly inactivated at 55°C
• Ideal for contamination control in RT-PCR
• Enables post-PCR analysis

Cod Uracil-DNA Glycosylase (Cod UNG) from Atlantic cod is the only commercially available UNG enzyme that is completely and irreversibly inactivated by moderate heat treatment. The enzyme is produced in recombinant E. coli (ung^–) strain that contains a modified Cod UNG gene.

There are several commercially available Uracil-DNA glycosylases on the market today. Most of them are of bacterial origin and work well if you have no intention to further analyze the PCR products post-PCR. However, if you want to store your PCR products for downstream analysis such as cloning and sequencing, the reactivation of UNG and subsequent degradation of your PCR products are a problem with most of the commercially available UNGs. Cod UNG from ArcticZymes is the only commercially available UNG today which is completely and irreversibly inactivated by heat. This is illustrated in Figure above, where various UNGs were tested for residual activity after heat inactivation. PCR was performed with dUTP and 1 Unit of 5 different commercially available UNGs. Post-PCR, the PCR products were incubated at room temperature for various time intervals, followed by heating and subsequent cooling. Gel electrophoreses of the PCR products revealed UNG reactivation, and thus severe degradation of PCR products of all UNGs tested, except for Cod UNG.

Recommended Protocol

One-step RT-PCR

• Add 0.2 U Cod UNG directly to your 20 µl RT-PCR reaction
• Preincubate at room temperature for 5 min
• Reverse transcribe your RNA at 50- 55°C
• Run your PCR

PCR

Cod UNG works in all commercially available master mixes. Be sure that you have used dUTP containing dNTP mixes in your previous PCR experiments.

• Add 0.25 U Cod UNG directly to your 25 µl PCR reaction
• Pre-incubate for 5 min at room temperature
• Run your PCR

Store your PCR product at -20°C or 4°C degrees for as long you want, before analysis.

Properties

Specifications

Unit definition: One Unit will liberate 1 nmol Uracil from Uracil-containing DNA per hour at 37°C.
Specific activity: >500 000 Units/mg.
Purity: Purified to apparent homogeneity by SDS-PAGE. No nuclease activity is detected.
Concentration: Minimum 1 000 Units/ml. (See below)

Stability

Minimum shelf life at -20°C is 2 years. In practice we find that storage at 4°C is possible for at least 6 months. The enzyme activity is also preserved upon multiple freeze-thaw cycles.

Note:

70500-201 concentration 1unit/ul  100ul = 100 units

70500-202 concentration 1unit/ul  1000ul =1000 units

70500-203 concentration 5unit/ul  500ul = 2500 units

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