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11
Protocol
1. Dissolve the DNA to be
labelled to a concentration
of 2.5–25 ng/µl in either
distilled water of 10 mM
Tris/HCl, pH8.0, 1 mM EDTA
(TE buffer).
Notes
1. If desired, 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.
5. Megaprime DNA labelling protocols
The Megaprime systems allow DNA from a variety of sources
to be labelled in vitro to high specific activity with
32
P and other
radionuclides. The specific activity of the probes generated by these
systems will vary according to the specific activity of the labelled
dNTP used.
The standard Megaprime protocol is presented, together with a new
protocol which reduces the variation in labelling efficiency that can
occur with DNA template from a variety of sources.
The protocols given here are for use with 17 pmol[α
–32
P]dNTP,
specific activity 3000 Ci/mmol. For alternative reaction conditions
refer to page 20.
DNA prepared by standard minilysate methods may be used in
either protocol. DNA solutions which are too dilute to be used
directly should be concentrated by ethanol precipitation followed by
redissolution in an appropriate volume of water or 10 mM Tris/HCl,
pH 8.0, 1 mM EDTA. DNA in restriction enzyme buffers may be added
directly to the reaction. The reaction can also be performed with
DNA in agarose gel slices (see note 3 and Appendix 1).
5.1. Standard Megaprime protocol