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Kansas State University

PREPARATION OF GENOMIC-BLOCKING DNA

  1. Add 10 M NaOH to the DNA sample (0.1-1 µg/µL) to a final concentration of 0.4 M NaOH.
  2. Place the DNA sample (in a microtube) in boiling water for 40 to 45 min.
  3. Cool the DNA sample on ice; add an equal volume of 3 M NaAc pH 4.6 and two volumes of 100% ethanol, and mix the sample well.
  4. Add a certain volume of TE, 1/10 volume of 3 M NaAc pH 4.6 and two volumes of 100% ethanol, mix the samples well, and repeat.
  5. Centrifuge for 10 min, drain tube, and rinse pellet and sides of the tube with 70% ethanol.  Drain the pellet well, dry, and dissolve in a certain volume of TE.
    • Other methods, such as autoclaving, shearing the DNA by passing it through a small needle of a syringe, or sonicating can be used to prepare blocking DNA. The advantage of the protocol described above is in the easy control of the size of the DNA ranging from 100 bp to 1 Kb, by boiling for 45 min. The DNA sample is clean because of repeated precipitations.