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31
Protocol
6. Continued.
The amount of radioactivity
incorporated during the
reaction (B) in dpm.
B = total number of µCi added x
2.2x10
4
x % incorporation
Thus the specific activity of the
labelled DNA is
specific activity = B x 10
3
dpm per µg
specific activity = A
Notes
B. Precipitation with trichloroacetic acid
Plastic or siliconized glass tubes must be used to avoid adsorption
of DNA.
1. Dilute an appropriate aliquot of the reaction mixture as described
in section A1.
2. Transfer 1–10 µl of diluted reaction mixture to two duplicate tubes
containing 200 µl water or 0.2M EDTA and 50 µl carrier DNA
solution. Mix well. Use this mixture (less any set aside in step 3) for
the TCA precipitation described in step 4 below.
3. Set aside an appropriate aliquot from each tube in step 2 for the
determination of total input radioactivity.
4. To the diluted samples from step 2, add 2 ml ice-cold 10%
trichloroacetic acid (TCA) solution, vortex, and allow to stand in an
ice-bath for 10–15 minutes. The labelled and carrier DNA will
co-precipitate. Note that TCA is corrosive, and care should be
taken in its handling.
5. Collect the precipitated DNA by vacuum filtration on a glass fibre
or nitrocellulose filter disc.