Cod Uracil-DNA Glycosylase
Cod Uracil-DNA Glycosylase
Brand: ArcticZymes
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Cod Uracil-DNA Glycosylase
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 a recombinant E. coli (ung-) strain that contains a modified Cod UNG gene.
Main advantages with Cod UNG
- Heat-labile
- Completely and irreversibly inactivated at 55°C
- Use of Cod UNG makes contamination control possible in RT-PCR
- Does not degrade PCR product post-PCR. This makes downstream use of the PCR product possible
- High purity enzyme, tested free of contaminating nucleases
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.
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.
Cod UNG is the only UNG compatible with one-step RT-qPCR
In reverse transcriptase qPCR (RT-qPCR), RNA is the initial template. However, the problem of carry-over contaminants can be as much of a problem here as in regular PCRs. Following reverse transcription, cDNA is the template for the PCR. If your sample is contaminated with carry-over PCR products from previous PCRs, the primers cannot distinguish between cDNA and carry-over DNA, resulting in erroneous results.
In one-step RT-qPCR kits, RNA is added to a master mix containing both reverse transcriptase and polymerase, allowing cDNA synthesis and qPCR in the same tube. E. coli UNG/UDG has an optimal working temperature up to approx. 50˚C and generally retains its activity up to approx. 70˚C. This temperature range is not compatible with carry-over prevention in one-step RT-qPCR protocols, as E.coli UNG/UDG would remove uracil incorporated into the cDNA during reverse transcription, thereby causing degradation of template.
Recombinantly expressed Cod UNG from ArcticZymes was originally isolated from the cold-adapted organism Atlantic cod. Cod UNG is highly active at temperatures ranging from 20˚C to 40˚C, quickly lose activity at temperatures above 42˚C and is irreversibly inactivated already at 55˚C. Since the optimum temperature range of Cod UNG is considerably lower compared to that of E. coli UNG/UDG, Cod UNG is compatible with use in single tube RT-qPCRs. Carry-over prevention is simply carried out by adding Cod UNG to a final concentration of 0.01 U/µl and introduce a 5 minute incubation step at 25˚C prior to initiation of RT-qPCR. Alternatively, the concentration can be increased to 0.04 U/µl and the pre-incubation step omitted. Cod UNG will efficiently remove carry-over contamination during sample setup and cycler ramping.
Here we show that Cod UNG can be used for carry-over prevention in a commercial one-step RT-qPCR master mix containing dUTPs instead of dTTPs. Treatment with Cod UNG did not affect target cDNA, yielding the same Cq-values as untreated samples. For comparison, treatment with a generic UNG resulted in significant Cq delay, demonstrating incompatibility with one-step RT-qPCR.
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Cod UNG is the only UNG compatible with one-step RT-qPCR
In reverse transcriptase qPCR (RT-qPCR), RNA is the initial template. However, the problem of carry-over contaminants can be as much of a problem here as in regular PCRs. Following reverse transcription, cDNA is the template for the PCR. If your sample is contaminated with carry-over PCR products from previous PCRs, the primers cannot distinguish between cDNA and carry-over DNA, resulting in erroneous results.
In one-step RT-qPCR kits, RNA is added to a master mix containing both reverse transcriptase and polymerase, allowing cDNA synthesis and qPCR in the same tube. E. coli UNG/UDG has an optimal working temperature up to approx. 50˚C and generally retains its activity up to approx. 70˚C. This temperature range is not compatible with carry-over prevention in one-step RT-qPCR protocols, as E.coli UNG/UDG would remove uracil incorporated into the cDNA during reverse transcription, thereby causing degradation of template.
Recombinantly expressed Cod UNG from ArcticZymes was originally isolated from the cold-adapted organism Atlantic cod. Cod UNG is highly active at temperatures ranging from 20˚C to 40˚C, quickly lose activity at temperatures above 42˚C and is irreversibly inactivated already at 55˚C. Since the optimum temperature range of Cod UNG is considerably lower compared to that of E. coli UNG/UDG, Cod UNG is compatible with use in single tube RT-qPCRs. Carry-over prevention is simply carried out by adding Cod UNG to a final concentration of 0.01 U/µl and introduce a 5 minute incubation step at 25˚C prior to initiation of RT-qPCR. Alternatively, the concentration can be increased to 0.04 U/µl and the pre-incubation step omitted. Cod UNG will efficiently remove carry-over contamination during sample setup and cycler ramping.
Here we show that Cod UNG can be used for carry-over prevention in a commercial one-step RT-qPCR master mix containing dUTPs instead of dTTPs. Treatment with Cod UNG did not affect target cDNA, yielding the same Cq-values as untreated samples. For comparison, treatment with a generic UNG resulted in significant Cq delay, demonstrating incompatibility with one-step RT-qPCR.
