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  • Antarctic Thermolabile UDG

    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.

    Product Source

    An E. coli strain that carries the cloned UDG gene from a psychrophilic marine bacterium

    Advantages and Features

    Applications

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

    Properties and 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, uracilcontaining 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 Temperature

    -20°C

    Storage Conditions

    20 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 min

    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.

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • DNase Activity (Labeled Oligo, 3' extension):
      The product is tested in a reaction containing a fluorescent  labeled double stranded oligonucleotide containing a 3' extension. The percent degradation is determined by capillary electrophoresis.
    • DNase Activity (Labeled Oligo, 5' extension):
      The product is tested in a reaction containing a fluorescent  labeled double stranded oligonucleotide containing a 5' extension. The percent degradation is determined by capillary electrophoresis.
    • Double Stranded DNase Activity (Labeled Oligo):
      The product is tested in a reaction containing a fluorescent  labeled double stranded oligonucleotide containing a blunt end. The percent degradation is determined by capillary electrophoresis.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • qPCR DNA Contamination (E. Coli Genomic):
      The product is screened for the presence of E. coli genomic DNA using SYBR® Green qPCR with primers specific for the E.coli 16S rRNA locus. Results are quantified using a standard curve generated from purified E. coli genomic DNA.  The measured level of E. coli genomic DNA contamination is less than 1 E.coli genome.
    • RNase Activity (Extended Digestion):
      The product is tested in a reaction containing a RNA substrate. After incubation for 16 hours greater than 90% of the substrate RNA remains intact as determined by gel electrophoresis.
    • Single Stranded DNase Activity (FAM Labeled Oligo):
      The product is tested in a reaction containing a fluorescent internal labeled single stranded oligonucleotide. The percent degradation is determined by capillary electrophoresis.

    Notes

    1. One unit of enzyme is capable of converting 2.3 nmol of 5´ FAM-labeled 26-mer ssDNA with a single uracil to 10-mer ssDNA in 30 minutes at 37°C following NaOH and heat treatment. Activity is performed in a 50 μl standard Taq reaction buffer containing 2 pmol of 5´ FAMlabeled 26-mer ssDNA with a single uracil and variable amount of enzyme in 30 minutes at 37°C.

    2. The NEB unit is 2–5 fold more active per unit than other suppliers. This Antarctic Thermolabile UDG is active in most PCR reaction buffers but is inhibited with increasing ionic strength (> 100 mM).

    References

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

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    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?