Richard Powell
Department of Microbiology, National University of Ireland, Galway, Ireland
Frank Gannon
European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, D-69012 Heidelberg,Germany
Equipment and reagents
Restriction endonucleases (enzymes) plus appropriate buffers
Water-bath or heating-block
Gel electrophoresis apparatus
5 M NaCl
Method
1 Make up a 100 l reaction mixturea by adding the reagents in order into a sterile 1.5 ml microfuge tube: 10 l 10× restriction endonuclease bufferb, x l DNA and y l water. The value of x will depend on the concentration of your DNA solutionc. The value of y should bring the volume up to 99 l. This reaction mixture should be appropriate for the restriction endonuclease that requires the lowest salt concentration when the two enzymes for use are compared. If the reaction buffers of both enzymes are similar, the DNA may simply be incubated in the presence of both enzymes.
