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18
Protocol
8. Incubate at 37°C for
10 minutes continued.
9. 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
8. Continued
When labelling DNA in low
melting point agarose,
longer incubation of 15–30
minutes at 37°C are
required for optimum
labelling. Longer incubation
times (up to 60 minutes)
are required when nucleotide
analogues (e.g. [
35
S]dNTP(S)
are used.
9. 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 unincorporated
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