Cryophile Uracil DNA Glycosylase
Cryophile Uracil DNA Glycosylase or Uracil-N glycosylase (UNG) hydrolyses the N-glycosylic bond between the deoxyribose sugar and the base in uracil- containing DNA leaving a basic (apyrimidinic) site in DNA. Cryophile Uracil DNA Glycosylase is not active on uracil in RNA. The high activity combined with a fast irreversible heat-inactivation step makes the enzyme ideal for contamination control in PCR and RT-PCR reactions. Unlike all other available UNG enzymes, Cryophile Uracil DNA Glycosylase does not reactivate after heat-inactivation. This enables contamination control in amplification reactions also where subsequent post-PCR analysis is required.
Features
- The only UNG that can be completely and irreversibly heat inactivated
- Heat-labile without any addition of agents or inhibitors
- Eliminates carry-over contamination in both PCR and RT-PCR
- Enables downstream post-PCR analysis such as cloning and sequencing
Source
Arctic cod origin, recombinantly produced in E. coli.
Heat inactivation
Cryophile Uracil DNA Glycosylase is completely and irreversibly inactivated by incubating the enzyme at 55°C for 20 min.
Storage
Minimum shelf life is 2 years at -20°C. Storage at 4°C is possible for at least 6 months. The enzyme also tolerates multiple freeze-thaw cycles.
Purity
Cryophile Uracil DNA Glycosylase highly pure and is tested free of contaminating nucleases.
Specific activity
> 500 000 Units/mg
Unit defnition
One unit of UNG is defined as the amount of enzyme required to release 1 nmol uracil from uracil-containing DNA per hour at 37°C
Use of Cryophile Uracil DNA Glycosylase in RT-PCR
A prerequisite for using UNG in RT-PCR contamination control, is that the enzyme is sufficiently heat-labile to quickly inactivate at the temperatures used for reverse transcription. The easily heat-inactivatable Cryophile Uracil DNA Glycosylase makes it possible to use contamination control also in RT-PCR, being able to remove more than 108 copies of contaminating DNA without affecting the sensitivity (Cq) of the assay. Applying Cryophile Uracil DNA Glycosylase in an RT-PCR reaction is done by a 5 min Cryophile Uracil DNA Glycosylase preincubation step at room temperature prior to the amplification reaction. Cryophile Uracil DNA Glycosylase does not affect sensitivity of qRT-PCR.
PCR product stability after Cryophile Uracil DNA Glycosylase treatment
The complete and irreversible heat-inactivation of Cryophile Uracil DNA Glycosylase enables the combination of contamination control and an array of post-PCR applications such as cloning, sequencing or genotyping. As Cryophile Uracil DNA Glycosylase is the only commercially available UNG that does not reactivate post-amplification, PCR products remain intact and can be stored for a prolonged period of time before further analysis.
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