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FAQs for DNA Modifying Enzymes and Cloning Technical Reference for DNA Modifying Enzymes and Cloning Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE Klenow Fragment (3´→5´ exo–) Octadeoxyribonucleotides in 10X Labeling Buffer

Home > Products > DNA Modifying Enzymes and Cloning > DNA Labeling and Chemiluminescent Detection > NEBlot Kit NEBlot Kit Catalog # Size Concentration Price Qty   N1500L 50 reactions   $193.00 N1500S 25 reactions   $110.00 Prices are in US dollars and valid only for US orders. Download: Technical Bulletin|Manual|MSDS PDF 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 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 Advantages: Produces high specific activity probes: 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. Kit Components: Control DNA dATP dCTP dGTP dTTP Klenow Fragment (3´→5´ exo–) Nuclease Free dH2O Octadeoxyribonucleotides in 10X Labeling Buffer Storage Conditions Storage Temperature: -20°C References Grunstein, M. and Hogness, D.S. (1975) Proc. Natl. Acad. Sci. USA, 72, 3961-3965. Southern, E.M. (1975) J. Mol. Biol., 98, 503-517. Smith, G.E. and Summers, M.D. (1980) Anal. Biochem., 109, 123-129. Hasse, A. et al. (1984) Methods of Virology, 7, 189-226. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem., 132, 6-13. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem., 137, 266-267. Reagents Sold Separately Klenow Fragment (3´→5´ exo–) Octadeoxyribonucleotides in 10X Labeling Buffer