Richard Powell
Department of Microbiology, National University of Ireland, Galway, Ireland
(richard.powell@nuigalway.ie)
Frank Gannon
European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, D-69012
Heidelberg, Germany (Frank.Gannon@EMBL-Heidelberg.de)
Equipment and reagents
T4 DNA ligase
10 × ligation buffer (0.66 M Tris-HCl, pH 7.6, 50 mM MgCl2, 50 mM DTT, 10 mM ATP)
Low temperature water-bath or heating-block
Gel electrophoresis apparatus
Method
1 The volume of the ligation mixture and the DNA concentration depend on the type of ligation experiment. Use a 10 l reaction with DNA at a concentration of <100 ng/l to promote the formation of concatamer ligation products, or a 10 l (or larger) reaction with DNA at a concentration of &10 ng/l to promote the formation of circular ligation products.
2 Add T4 DNA ligase. Check the supplier’s documentation on enzyme activity, or more generally, add 0.25 units enzyme/g of DNA for cohesive-end ligations, and 2.5 units/g of DNA for blunt-end ligations.
3 Incubate the reaction mixture at 15 °C for 1–16 h. Simple cohesive-end ligations are usually complete in 1 h.
4 Analyse for correct and complete ligation by gel electrophoresis using unligated material as a suitable control, or also by transformation of E. coli cells if appropriate (i.e. the ligation includes an appropriate plasmid vector).
