DNA ligation

2010-03-05 16:49 · Ernest

Richard Powell Department of Microbiology, National University of Ireland, Galway, Ireland (richard.powell@nuigalway.ie) Frank Gannon Eu

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).

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