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16
Protocol
3. Place 25 ng (5 µl) of
template DNA into a clean
microcentrifuge tube and to
it add 5 µl of primers.
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 10x
reaction buffer (RPN 1604/5)
or the labelling buffer (RPN
1606/7), water and enzyme:-
Component RPN1604/5 RPN1606/7
Labelling 10 µl
buffer
Unlabelled 4 µl of each –
dNTPs omitting
those to be
used as label
Notes
3. If the volume of DNA and
primers is less than 10 µl
make up to this volume with
water. 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 enzyme can be added
directly to the reaction mix
or pipetted on to the side of
the microcentrifuge tube
and “washed” down with the
water.