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  • Recommended Protocol for 3H label of DNA

    Protocol

    1. Obtain tritiated SAM: NEB recommends GE Healthcare Life Sciences S-adenosyl-L[methyl-3H]methionine; Code TRK236, 15 Ci/mmol which is about 66 μM.
    2. Add in order:
      a. Nuclease free water 12 μl
      b. 10X NEBuffer 2 2 μl
      c. Add SAM from step 1 4 μl
      d. DNA (1 mg/ml) 1 μl
      e. SssI methylase (4 U/μl) 1 μl
    3. Mix, Pipette up and down at least six times.
    4. Incubate one hour at 37°C.
    5. Stop the reaction by heating at 65°C for 20 minutes.

      Notes:

      1. The volume of DNA can be increased to 5 μl. When using more dilute DNA increase the reaction volume to 50 μl. Using too much DNA volume in the reaction can cause inhibition by changing the pH or salt concentration of the reaction.
      2. The volume of SAM can be increased to 8 μl without inhibition. If more label is required, larger volumes of SAM can be dried in a spin vacuum. The reaction is then set up using the tube containing dried SAM.
      3. The incubation time can be increased to 4 hours. Overnight incubations do not give significant increases in methylation.
      4. Using a similar protocol we were able to label 1 μg of lambda DNA to 3.43 x 106 cpm measured by a standard filter-binding assay. Quenching in the assay was about 40% (D. Robinson unpublished observation).