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  • NEBlot® Kit

    Description

    The NEBlot® Kit is designed to produce labeled DNA probes of high specific activity suitable for use as hybridization probes for screening gene libraries (1), probing of Southern and Northern blots (2,3) and in situ hybridization (4). 

    Using the method of Feinberg and Vogelstein (5,6), random sequence octadeoxyribonucleotides serve as primers for DNA synthesis in vitro from denatured, double-stranded template DNA by the Klenow Fragment of E. coli DNA Polymerase I. 

    The method permits the labeling of small quantities of DNA (< 25 ng). The resulting extremely efficient use of labeled deoxynucleotide triphosphate allows probes of high specific activity to be produced. It also enables the labeling of DNA in the presence of low-melting point (LMP) agarose, thus overcoming the major limitations of earlier nick translation methodology. 

    The system produces consistent results; typically, probes labeled to > 1 x 109 dpm/µg can be achieved within 30 minutes using quantities of DNA from 25 ng to 2 µg. All four dNTPs are provided allowing the researcher flexibility in choosing label. The performance of the system may be monitored by labeling the control DNA provided with the system. 

    NEBlot Kit is tested for its ability to label 25 ng of HindIII digested lambda DNA to a specific activity of > 1 x 109 dpm/µg.

    Incorporation of Label at Varying Template Concentrations
    Preparation of Probes Using the NEBlot Kit

    Highlights

    • Produces high specific activity probes
    • Direct labeling of DNA in the presence of low-melting point (LMP) agarose
    • All four dNTPs are provided. This allows the researcher flexibility in choosing label (labeled nucleotide triphosphates are not supplied) System performance monitored by labeling of control DNA
    • The NEBlot Kit is tested for its ability to label 25 ng of HindIII digested lambda DNA to a high specific activity (>1 x 109) and is guaranteed for one year from the date of assay

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    dATP Solution-200.5 mM
    dCTP0.5 mM
    dGTP0.5 mM
    dTTP0.5 mM
    Octadeoxyribonucleotides in 10X Labeling Buffer400 μg/ml
    Control DNA, HindIII digested lambda DNA25 μg/ml
    Nuclease Free dH2O
    Klenow Fragment (3'→5' exo-)-205,000 units/ml

    Advantages and Features

    Features

    • The efficient incorporation of dNTPs allows probes of a very high specific activity to be produced. Typically, probes of a specific activity of >1 x 109 dpm/µg can be prepared within 30 minutes, beginning with template DNA levels ranging from 25 ng to 2 µg.
    • Direct labeling of DNA in the presence of low-melting point (LMP) agarose: 
    • Enables probes to be easily prepared from a restriction fragment isolated by agarose gel electrophoresis. This overcomes the major limitations of earlier nick translation methodology; i.e., relatively low levels of label incorporation, the necessity for large quantities of template DNA (nearly 1 µg), and little flexibility of reaction conditions.

    Properties and Usage

    Storage Temperature

    -20°C

    Shipping Notes

    • Ships on dry ice

    References

    1. Grunstein, M. and Hogness, D.S. (1975). Proc. Natl. Acad. Sci. USA. 72, 3961-3965.
    2. Southern, E.M. (1975). J. Mol. Biol.. 98, 503-517.
    3. Smith, G.E. and Summers, M.D. (1980). Anal. Biochem.. 109
    4. Hasse, A. et al. (1984). Methods of Virology. 7
    5. Feinberg, A.P. and Vogelstein, B. (1983). Anal. Biochem.. 132
    6. Feinberg, A.P. and Vogelstein, B. (1983). Anal. Biochem.. 137, 266-267.

    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.

    Manuals

    The Product Manual 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 these document files: manual[Catalog Number].
    1. Can Klenow Fragment (3'→5' exo-) be used in labeling reactions and partial fill in reactions?
    2. Is nick translation the best way to make a labeled probe?
    3. Is SP6 RNA Polymerase an enzyme of choice for making high specific activity labeled probes?
    4. Is T7 RNA Polymerase an enzyme of choice for making high specific activity labeled probes?
    1. Determination of % Incorporation of Label into probe generate by the NEBlot Kit
    2. Purification of labeled probe generate form the NEBlot Kit
    3. Generation of Labeled Probe using the NEBlot Kit
    In order to minimize radiolysis, it is strongly recommended to use the radioactive probes promptly. Store the kit at -20 and avoid repeated freezing and thawing.