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Kinase Reaction

Protocol

  1. Add 4 μl of linker.
  2. Add 6.6 pmoles (20 μCi) of [γ- 32P]ATP (sp. act. &#gt; 3,000 Ci/mmole).
  3. Add 1 μl of 10X kinase buffer (700 mM Tris-HCl (pH 7.6), 100 mM MgCl2, 50 mM DTT).
  4. Add water to 9 μl.
  5. Add 1 μl T4 Polynucleotide Kinase.
  6. Incubate at 37°C for 15 minutes.
  7. Add 1μl 10X kinase buffer, 1 μl 10 mM ATP, 7 μl H20, 1 μl T4 Polynucleotide Kinase. Incubate at 37°C for 30 minutes.
  8. Heat inactivate reaction 65°C for 20 minutes.