A Typical DNA Tailing Reaction

Protocol

  1. Mix: a. 5.0 μl 10X TdT Buffer
    b. 5.0 μl 2.5 mM CoCl2 solution provided
    c. 5.0 pmols DNA (330 ng for 100 bp, 1 µg for 300 bp, 10 pmols DNA ends)*
    d. 0.5 μl 10 mM dNTP (alpha-32P dATP may also be used)
    e. 0.5 μl Terminal Transferase (20 units/μl) deionized H20 to a final volume of 50 μl.

    *To determine approximate amount of DNA (ng/pmol), multiply the number of base pairs by 0.66. Example: 300 bp x 0.66 = 198 ng/pmol. For 5.0 pmols multiply by 5, resulting in 990 ng/5 pmol.

    The table below can be used as a guide (values are approximate and are given for a 30 minutes incubation at 37°C in the recommended buffer).

    The rate of addition of dNTP's and thus the length of the tail is a function of the ratio of 3´ DNA ends: dNTP concentration, and also which dNTP is used.

    DNA Tailing Guide:

    pmols 3' ends
    pmols dNTP
    Tail Length 
      dA dC dG dT
    1:100  1-5  1-3  1-3  1-5
    1:1,000  10-20  10-20  5-10  10-20
    1:5,000  100-300  50-200  10-25  200-300

  2. Incubate at 37°C for 30 minutes.
  3. Stop the reaction by heating to 70°C for 10 minutes or by adding 10 µl of 0.2 M EDTA (pH 8.0).