15
Protocol
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
continued.
Notes
8. Continued
under the conditions given
above is not required with
the isotopes
32P and 33P.
Purification of
35S labelled
probes is however required
to reduce filter background.
5.2. New Megaprime protocol
Protocol
1. Dilute the DNA to a
concentration of 5 ng/µl in
either distilled water or
10 mM TE buffer.
2. Place the required tubes
from the Megaprime system
with the exception of the
enzyme at room temperature
to thaw. Leave the enzyme
at -15°C to -30°C until
required, and return
immediately after use.
Notes
1. DNA solutions at
concentrations in the range
5–25 ng/µl can be used if desired.
However the denaturing
volume (step 3) should not be
less than 10 µl to maximize
the efficiency of primer
annealing. The labelling
efficiency of a DNA sample
can be compared with that
of the standard DNA supplied
with the kit. In this case 5 µl
of standard DNA should be
used.