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Protocol
3. Add water to a ratio of 3 ml
per gram of gel and place
in a boiling water bath for
5 minutes to melt the gel and
denature the DNA.
4. If the DNA is to be used
immediately remove the
appropriate volume
containing 25 ng, add to
the primers as indicated in
the labelling protocol (page
11, step 3). The volume of
DNA should not exceed 25 µl
for the standard labelling
protocol.
5. Incubate the labelling
reaction for 15–20 minutes
at 37°C.
Notes
3. If the DNA is not to be used
immediately divide the boiled
samples into suitably sized
aliquots and store at -15°C to
-30°C in a non frost-free
freezer.
4. When using DNA which has
been previously boiled and
then stored at -15°C to -30°C,
first place the tube in a
boiling water bath for
30 seconds to melt the
agarose, before removing the
required volume containing
25 ng. Do not reboil DNA
aliquots more than twice.
6.2. Appendix II. Monitoring the reaction and
calculating the specific activity of the labelled
DNA
A. Adsorption to DE81 paper
Monitoring of the progress of the labelling reaction and
measurement of probe specific activity can be achieved by
determining the proportion of the radionucleotide incorporated
during the Megaprime reaction.