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  • Generation of Labeled Probe using the NEBlot Kit

    Overview

    Choice of Label:
    NEBlot® Kit can be used with the labeled nucleotide of your choice.  Listed below are guidelines for the use of some common nucleotide triphosphates used in labeling reactions.

    Random Priming Reaction:
    Allow system components to thaw on ice. Klenow enzyme, however, should be kept at –20°C except when being used.

    Standard 50 μl reaction using 32P dCTP

    For a standard 50 μl reactoin, total volume of DNA and label should be 38 μl.

    Protocol

    1. In a microcentrifuge tube:
      Dissolve 25 ng of template DNA in nuclease free H2O (1-33 μl).
      For the control reaction, add 1μl of control DNA to 32 μl of H2O.
    2. Denature in boiling H2O bath for 5 minutes (see manual Figure 1, page 7).
    3. Quickly place in ice for 5 minutes.
    4. Centrifuge briefly in the cold.
    5. Add the following reagents to your DNA in the order listed.

      • 5 μl Octadeoxyribonucleotides in 10X Labeling Buffer
      • 6 μl dNTP mixture (2 μl of dATP, dTTP, and dGTP).
      • 5 μl α 32P dCTP (3,000 ci/mmol, 50 μCi)
      • 1 μl DNA Polymerase I – Klenow Fragment (3’ → 5’ exo-) (5 units)
    6. Incubate at 37oC for 1 hour (see manual Figure 1, page 4 for other labels).
      Incubation time for DNA/Agarose 6 hours for 32P [ ]
    7. Terminate reaction by adding 5 μl of 0.3 M EDTA (pH 8.0).

      We recommend purification of probes to reduce non-specific background and limit exposure of lab personnel to high levels of radioactivity during hybridization experiments.