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30
Protocol
5. Place the squares in separate
vials with at least 5 ml of
scintillation fluid and count.
6. Efficiency of counting will
vary, but the percentage
incorporation can be used
to calculate probe specific
activity. Unlike the nick
translation labelling reaction,
Megaprime labelling leads
to net DNA synthesis, and
so the total amount of DNA
at the end of the reaction
must be calculated.
Total amount of DNA (A) ng =
Total number of µCi added x 13.2* x % incorporation + 25
Number of radioactive dNTPs added x average specific
activity of dNTPs added
This assumes a 25% content
of any one dNTP in the newly
synthesized DNA, and
25 ng of template DNA.
*13.2 equals four times the
average molecular weight of
the four dNTPs divided by 100.
Notes
5. Determination of the
proportion of the
32
P labelled
nucleotide incorporated may
be achieved using Cerenkov
counting if desired in this
case drying the filter is not
necessary.
6. The mean value of the
counts on the washed filter
represents the proportion
of the radionucleotide
incorporated into the DNA
probe, while the mean of the
unwashed filters represents
the total amount of
radioactivity in the reaction
mix, such that;
% incorporation = mean counts on washed filters x 100
mean counts on unwashed filters