17
Protocol
Reaction 5 µl –
buffer
Enzyme 2 µl 2 µl
Water* as appropriate
for a final
reaction
volume of
50 µl*
* When calculating this volume
remember to allow for the
volume of radioactive
nucleotide to be added.
6. Cap the tube and spin for a
few seconds in a
microcentrifuge to bring the
contents to the bottom of the
tube.
7. Add the radiolabelled dNTP,
for example 5µl [α–32P]dNTP,
specific activity 3000 Ci/mmol.
Mix by gently pipetting up and
down. Spin for a few seconds
in a microcentrifuge to bring
the contents to the bottom of
the tube.
8. Incubate at 37°C for
10 minutes.
Notes
7. Avoid vigorous mixing of the
reaction mixture as this can
cause severe loss of enzyme
activity.
8. Purified DNA can be labelled
to high specific activity in
10 minutes at 37°C but, if
desired can be labelled for
up to 1 hour at this
temperature.