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14
Protocol
7. Incubate at 37°C for
10 minutes continued.
8. Stop the reaction by the
addition of 5 µl of 0.2 M EDTA.
For use in a hybridization,
denature the labelled DNA
by heating to 95–100°C for
5 minutes, then chill on ice.
Notes
7. Continued.
times (up to 60 minutes)
are required when nucleotide
analogues (e.g. [
35
S]dNTPαS)
are used.
8. Labelled probe can be stored
at -15°C to -30°C in a non
frost-free freezer. Prolonged
storage of
32
P-labelled
probes can lead to substantial
probe degradation(7). High
specific activity probes
should be stored for no
longer than 3 days. Although
probe purification is not
usually necessary for most
membrane applications,
the removal of unicorporated
nucleotide is sometimes
useful to reduce background
in filter hybridizations
for probes >10
9
dpm/µg or
when the reaction yields an
incorporation of less than 50%.
This procedure is described
in Appendix III. Calculation
of probe specific activity
is described in Appendix II.
Extensive experimentation
with Rapid-hyb buffer
(RPN1635/6) has shown that
probe purification, even