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Protocol
1. Fractionate restriction
endonuclease digested DNA
in a suitable low melting
point agarose gel containing
0.5 µg/ml ethidium bromide.
Estimate the DNA content
of the band by reference to
a set of standards of known
concentration on another
track. 250 ng should allow
25 ng to be used in the
standard labelling protocol
without further concentration
2. Excise the desired band
cleanly, with the minimum
of excess agarose and
transfer to a pre-weighed
1.5 ml microcentrifuge tube.
Notes
1. A low melting point agarose
of high purity for example
SepRate-LMP is
recommended for maximum
labelling efficiency.
2. It is recommended that
the exposure to UV light
is minimized, as prolonged
exposure can damage the
DNA.
6. Appendices
6.1. Appendix I. Labelling of DNA fragments in
low melting point agarose
The DNA samples produced by the following protocol have been
found to be labelled to approximately the same extent as purified
DNA. 15–20 minutes at 37°C is optimum for labelling. The standard
labelling protocol may be found to be more appropriate for labelling
DNA in agarose as the volume of DNA to be added using the new
protocol is limited to 5 µl, requiring a relatively high initial DNA
concentration.