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Antarctic Thermolabile UDG (Uracil-
DNA Glycosylase) catalyzes the release of free
uracil from uracil-containing single-stranded or
double-stranded DNA. The resulting abasic sites
are susceptible to hydrolytic cleavage at elevated
temperature and high pH. This enzyme is sensitive
to heat and can be rapidly and completely inactivated
at temperatures above 50°C.
Figure 1: Evaluation of RT-qPCR carryover prevention
To evaluate the capacity of carryover product digestion, a uracil-containing RT-qPCR product was generated using the Luna One-Step Probe RT-qPCR kit. Approximately 105 copies of the uracil-containing product were used as template for subsequent qPCR reactions using three different RT-qPCR master mixes (all containing UDG). Following manufacturer’s recommended protocols, incubation at 25˚C at the start of the 2nd reaction enables UDG to degrade the dU-containing input, reducing its ability to contribute to a (false) positive signal. The ΔCq value is the cycle difference between carryover treatment and no carryover treatment of the same input. Larger ΔCq values indicate more efficient carryover product digestion. After decontamination using the Luna mix, full product digestion was observed in one sample and a large ΔCq was observed in the second. Note that the amount of DNA present in true contamination events is difficult to assess/predict.
Product Source
An E. coli strain that carries the cloned UDG gene from a psychrophilic marine bacterium
This product is related to the following categories:
One unit is defined as the amount of enzyme that catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracil-containing DNA. Activity is measured by release of [3H]-uracil in a 50 μl Standard Taq Reaction Buffer containing 0.2 μg DNA (104–105 cpm/μg) in 30 minutes at 37°C.
Reaction Conditions
1X Standard Taq Reaction Buffer Pack
Incubate at 37°C
1X Standard Taq Reaction Buffer Pack
10 mM Tris-HCl
50 mM KCl
1.5 mM MgCl2
(pH 8.3 @ 25°C)
Storage Buffer
10 mM Tris-HCl
50 mM NaCl
0.1 mM EDTA
1 mM DTT
50% Glycerol
pH 7.5 @ 25°C
Heat Inactivation
50°C for 5 minutes
Unit Assay Conditions
1X Taq Reaction Buffer, 1 unit of uracil DNA Glycosylase, 0.2 μg 3H-uracil DNA (104 –105 cpm/μg) for 30 minutes at 37°C in a total reaction volume of 50 μl.
One unit of enzyme is capable ofconverting 2.3 nmol of 5´ FAM-labeled 26-merssDNA with a single uracil to 10-mer ssDNA in30 minutes at 37°C following NaOH and heattreatment. Activity is performed in a 50 μl standardTaq reaction buffer containing 2 pmol of 5´ FAMlabeled26-mer ssDNA with a single uracil andvariable amount of enzyme in 30 minutes at 37°C.
The NEB unit is 2–5 fold more active per unit thanother suppliers. This Antarctic Thermolabile UDG isactive in most PCR reaction buffers but is inhibitedwith increasing ionic strength (> 100 mM).
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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