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c. Use of [
32
P]dNTPαS.
When using
32
S-labelled radionucleotides the incubation time should
be extended to 1 hour at 37°C.
d. Labelling at room temperature.
If desired, labelling reactions can be carried out at room
temperature. Maximum incorporation occurs after an incubation
time of 45–60 minutes. A decline in incorporation can be observed if
reactions are left overnight.
e. Factors affecting the labelled DNA.
1. Specific activity
Figure 3a should be used to ascertain the number and quantity of
labelled dNTP’s required in order to prepare a probe of the desired
specific activity.
2. Efficiency
Figure 3b indicates the efficiency of the chosen reaction
conditions, and thus permits a balance of specific activity and
economy.
3. Probe length
Figure 3c gives a measure of mean probe lengths obtained under
standard conditions. Probe lengths were measured by denaturing
agarose gel electrophoresis followed by autoradiography with
reference to molecular weight standards.
Probe length can be affected by the concentration of DNA, primer
and nucleotide, the size of the template DNA and also radiolysis
of the labelled probe. The data in the figure was obtained using
linearized plasmid DNA, 4.5 Kb in length under the standard
labelling conditions.
It is recommended that not less than 10 pmol and not more
than 125 pmol of any labelled dNTP is used in the reaction and
combinations shown offer optimum balance of stability, specific
activity and economy.
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