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  • β-Agarase I

    Description

    β-Agarase cleaves the agarose subunit, unsubstituted neoagarobiose [3,6-anhydro-a-L-galactopyranosyl-1-3-d-galactose] to neoagaro-oligosaccharides (1).

    Highlights

    • Isolated from a recombinant source
    • DNase and RNase Free
    • Supplied with 10X Reaction Buffer

    Product Source

    Isolated from a strain of E. coli that carries a plasmid which encodes the β-Agarase I gene.

    Advantages and Features

    Applications

    • β-Agarase I digests agarose, releasing trapped DNA and producing carbohydrate molecules which can no longer gel. β-Agarase I can be used to purify both large (> 50 kb) and small (< 50 kb) fragments of DNA from gels. The remaining carbohydrate molecules and β-Agarase I will not, in general, interfere with subsequent DNA manipulations such as restriction endonuclease digestion, ligation, and transformation.

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to digest 200 μl of molten low melting point or NuSieve agarose to nonprecipitable neoagaro-oligosaccharides in 1 hour at 42°C.

    Usage Concentration

    1%

    Storage Temperature

    -20°C

    Storage Conditions

    50 mM Bis-Tris-HCl
    1 mM EDTA
    50% Glycerol
    pH 6.5 @ 25°C

    Heat Inactivation

    65°C for 15 min

    Quality Control

    Quality Assurance Statement

    • Purified free of contaminating endonucleases, exonucleases and ribonucleases.

    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.
    • Endonuclease Activity (Nicking, Buffer):
      The buffer 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.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. β-Agarase I retains activity for several hours at 40-45°C and is stabilized by the presence of agarose in the reaction.
    2. Only low melting point agarose is suitable for β-Agarase I digestion as the solution must be liquid at the incubation temperature of 42°C. If the temperature falls below 42°C during the reaction time, even low melting point agarose will begin to congeal and be undigestable.
    3. β-Agarase I is quickly inactivated at temperatures above 45°C. Therefore, when working with large volumes, be sure to leave ample time for the molten agar to equilibrate to 42°C.
    4. β-Agarase I works best on gels made with Tris-acetate buffer (TAE). For gels made with Tris-borate buffer (TBE), doubling the required amount of β-Agarase I is recommended.
    5. β-Agarase I works most efficiently on solutions containing 1% agarose or lower. For maximum digestion of higher percentage gels, melt the gel slice at 65°C and adjust the volume with 1X β-Agarase I Buffer so that the final concentration of agarose is 1%.
    6. β-Agarase I exhibits optimal activity at pH 6.5. Greater than 75% of the optimal activity is maintained between pH 5.0-8.5.
    7. Incubation at 95°C for 2 minutes or incubation at 65°C for 15 minutes inactivates 50 units of β-Agarase I. β-Agarase I retains activity for several hours at 40°-45°C and is stabilized by the presence of agarose in the reaction.

    References

    1. Yaphe, W. (1957). Can. J. Microbiol.. 3, 987-993.
    2. Davis, T. and Guan, C. Unpublished observation

    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. Can ligation and other DNA manipulations be carried out on β-Agarase I treated gel slices containing DNA?
    2. What is the molecular weight of β-Agarase I?
    3. What type of agarose will β-Agarase I digest?
    4. Will β-Agarase I work in other buffers?
    5. Why does a white precipitate form after the reaction using β-Agarase I?
    6. Can a high percentage low melt gel be digested with β-Agarase I?
    7. What is a common cause of β-Agarase I reaction failure?
    8. Does the gel running buffer have an effect on the β-Agarase I reaction?
    9. How can β-Agarase I be heat inactivated?
    10. How stable is β-Agarase I in reaction?
    11. What is the optimal pH for β-Agarase I digestion?
    1. DNA Purification
    2. Agarose Digestion