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  • Determination of % Incorporation of Label into probe generate by the NEBlot Kit

    Protocol

    1. Remove a 1 μl sample from the reaction mixture and dilute to 100 μl with 0.2 M EDTA.

    2. Spot 5 μl of the diluted sample onto each of two glass fiber filters, nitrocellulose discs, or 3mm paper discs.
      One of the filters is used to measure total activity in the reaction.  The other filter is used to measure only acid-precipitable radioactivity.  Under these conditions DNA more than 50 nucleotides long will be precipitated on the surface of the filter.

    3. Add one filter to a beaker containing 100 ml of ice-cold 5% Trichloroacetic acid (TCA) and 1% Sodium Pyrophosphate.  Let sit for 10 minutes.
      TCA is extremely corrosive and should be handled carefully.

    4. Wash two times with fresh, cold TCA/Pyrophosphate solution.

    5. Remove the filter from the TCA/Pyrophosphate solution.  Place the filter in a beaker containing 200 ml of ethanol or isopropanol and agitate briefly.

    6. Remove the filter from the alcohol and dry both filters under a heat lamp for 15 minutes.

    7. Place each of the filters (washed and unwashed) into scintillation vials.

    8. Measure the radioactive decay as shown below.

    9. Absorption to DE-81 Filter Method
      Remove a 1 μl sample from the reaction mixture and dilute to 100 μl with 0.2 M EDTA.

    10. Spot 5 μl of the diluted sample onto each of two DE-81 (2.4 cm) filters.
      One of the filters is used to measure total radioactivity in the reaction and is not subsequently washed.  The other filter is used to measure radioactivity incorporated.

    11. Dry the filters under a heat lamp for 15 minutes.

    12. Add one filter to a beaker containing 100 ml of 0.5 M Na2HPO4.  Wash for 5 minutes using mild agitation.  Wash two additional times with fresh 0.5 M Na2HPO4.

    13. Next, wash the filter twice with dH2O the repeat wash two times with ethanol.

    14. Dry filter uner a heat lamp for 15 minutes, then place in scintillation vial.

    15. Measure Radioactive decay as shown below.

    16. Measurement of Radioactive Decay
      For measurement of radioactive decay, use one of the following methods:

      A. Dissolve the nitrocellulose filters in 10 ml of toluene based liquid scintillation cocktail.
      B. Add 10 ml of a toluene based liquid scintillation cocktail directly to the glass fiber filter or paper filter and count using the appropriate channel of the liquid scintillation counter. 
      C. To measure radioactivity directly from the dry filters (Cerenkov counting) use the tritium channel of the liquid scintillation counter to measure 32P decay. 

      For calculation of Probe Specific Activity, please see product manual.