Product Class: Other

Antarctic Thermolabile UDG
NEBU cloned at NEB recombinant 37 50 Heat

Product Introduction

  • Uracil DNA glycosylase
  • Monofunctional DNA glycosylase that catalyzes the hydrolysis of the N-glycosidic bond from deoxyuridine to release uracil
  • Active on ss and dsDNA
  • Can be completely inactivated at temperatures above 50°C
  • Component of Thermolabile User II Enzyme (NEB #M5508)
  • Helps to prevent carryover contamination in LAMP (NEB #M1804, NEB #M1708) and qPCR-related technologies (Luna® Probe One-Step RT-qPCR 4X Mix with UDG (NEB  #M3019))
Catalog # Size Concentration
M0372S 100.0 units 1000 units/ml
M0372L 500.0 units 1000 units/ml

Product Information

Description

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:
DNA Repair Enzymes and Structure-specific Endonucleases Products
This product can be used in the following applications:
Loop-Mediated Isothermal Amplification

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

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.

Application Features

  • Prevention of Carry-over Contamination in PCR (1)
  • Remove Uracil-base from DNA

Product Notes

  1. 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.

  2. 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).

References

  1. Longo, M.C. et al. (1990). Gene. 93, 125-128.

Protocols, Manuals & Usage

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the activity of UDG in the NEBuffers 1-4?
  2. Will UDG work in T4 DNA ligase buffer?
  3. What is the molecular weight of the thermolabile UDG?
  4. Is UDG a tagged protein?
  5. I see that the Antarctic Thermolabile UDG works at 25-37°C. Do you have another glycosylase that works at higher temperatures?
  6. Does UDG release uracil from ss and dsDNA?
  7. Does UDG cut RNA?
  8. Can UDG be used to remove dU from a short 21mer oligo? Do the dU residues need to be spaced in any special way to avoid problems with cleavage?
  9. Are there any specific recommendations for the use of UDG on single stranded DNA? The materials on the web and data card seem to describe conditions for use with dsDNA.
  10. Will Antarctic Thermolabile UDG cleave uracil-containing DNA in a form of RNA-DNA duplex?
  11. What is the difference between UDG and UNG?
  12. How much Antarctic Thermolabile UDG I should add in a PCR reaction to prevent carry-over contamination?
  13. How do I use Antarctic Thermolabile UDG for carryover prevention in LAMP reactions?

Quality, Safety & Legal

Quality Assurance Statement

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.

Specifications

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]

Certificate Of Analysis

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]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

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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.