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12
Protocol
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.
3. Place 25 ng of template DNA
into a microcentrifuge tube
and to it add 5 µl of primers
and the appropriate volume
of water to give a total
volume of 50 µl in the final
Megaprime reaction.
Denature by heating to
95–100°C for 5 minutes
in a boiling water bath.
4. Spin briefly in a microcentrifuge
to bring the contents to the
bottom of the tube.
5. Keeping the tube at room
temperature, add the
nucleotides and reaction
buffer (RPN 1604/5) or the
labelling buffer (RPN 1606/7)
followed by the radiolabelled
dNTP(s) and enzyme as
follows:
Notes
3. When labelling DNA in low
melting point agarose, first
place the tube containing the
stock DNA in a boiling water
bath for 30 seconds to melt
the agarose before removing
the required volume. The
volume of low melting point
agarose DNA should not
exceed 25 µl in a 50 µl reaction.
5. The reaction volume may be
scaled up or down if more or
less than 25 ng of DNA is to
be labelled.