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  • RecJf

    Description

    RecJf is a single-stranded DNA specific exonuclease that catalyzes the removal of deoxy-nucleotide monophosphates from DNA in the 5' → 3' direction (1). RecJf is a recombinant fusion protein of RecJ and maltose binding protein (MBP). It has the same enzymatic properties as wild-type RecJ. Fusion to MBP enhances RecJf solubility (2).

    When DNA containing a 22 base 5' extension is used as a substrate for RecJf, the resulting products are a mixture of DNA fragments that have blunt-ends, 5' extensions (1-5 nucleotides) and recessed 5' ends (1-8 nucleotides) (3). RecJf does not require a 5' phosphate (3).

    Highlights

    Isolated from a recombinant source
    Supplied with 10X Reaction Buffer
    5' -> 3' exonuclease

    Product Source

    RecJf is overexpressed and purified as a C-terminal fusion to maltose-binding protein (MBP). MBP does not affect the catalytic activity of RecJf but does enhance RecJf solubility (2).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to produce 0.05 nmol TCA soluble deoxyribonucleotide in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X NEBuffer 2 with 1.5 µg sonicated single-stranded [3H]-labeled E. coli DNA.

    Reaction Conditions

    1X NEBuffer 2
    Incubate at 37°C

    1X NEBuffer 2:
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    1 mM DTT
    0.1 mM EDTA
    200 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Quality Control

    Quality Assurance Statement

    • Purified free of endonucleases and contaminating exonucleases.

    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.
    • Endonuclease Activity (Circular Single Stranded DNA):
      The product is tested in a reaction containing circular single stranded DNA. After incubation the percent cleaved is determined by agarose gel 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.

    References

    1. Lovett, S.T., Kolodner, R.D. (1989). Proc. Natl. Acad. Sci. USA. 86, 2627-2631.
    2. Lovett, S. and Whitaker, R. Unpublished observation
    3. Whitaker, R. 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. What is the activity of RecJf in the NEBuffers?
    2. Can DNA be blunted using RecJf?
    3. What is the difference between RecJf and Exonuclease I (NEB# M0293)?
    4. Can RecJf be heat inactivated?

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