of DNA with Restriction Enzymes
Heslop-Harrison, Molecular Cytogenetics, University of Leiceste. E-mail: PHH4 (a) le.ac.uk
of reaction is convenient for electrophoresis in agarose gel, but scale
can be adjusted.
the following into a 1.5 ml clean microcentrifuge tube on ice
: DNA in TE buffer or water (typically 5 to 50 micrograms); measure the DNA concentration by spectrophotometry)
restriction buffer (use corresponding buffer
to the enzyme, e.g., for HindIII,
use Buffer B(Roche) or NE Buffer 2 (BioLab))
H2O to 20 µl
(high quality water , e.g.,purchased from Sigma)
restriction endonuclease and incubate overnight (or at least 3 hr) at
DNA should be digested completely. Crude DNA preparations may require
more enzyme. The volume of enzyme should not exceed 1/10 the volume
of the final reaction mixture, because glycerol in the enzyme storage
buffer may interfere with the reaction.
the reaction and prepare it for electrophoresis by adding 2 µl
(10% of reaction vol) of 10×
on agarose gel, stain with ethidium bromide and photograph. Select agarose
content (see below). For Southern blotting 0.5 % agarose gel is recommended
and run slowly with 5 V per cm gel length (e.g. 50V for 10 cm gel).
For most applications, we prefer TBE buffer (more buffering capacity
but more expensive than TAE).
agarose concentrations for separating DNA fragments of various sizes
range of resolution of linear
DNA fragments (kb)
up to 7% for resolution under 100 bases, but note the cost is >£5 $USD10
per gel, and acrylamide
electrophoresis may be more appropriate