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
Reagents
Taq DNA polymerase
10 × Taq DNA polymerase buffer (200 mM Tris-HCl, pH 8.5, 15 mM MgCl2, 500 mM KCl)
Stock solution (e.g. 20 mM) of dTTP
T4 DNA ligase and 10 × ligation buffer (0.66 M Tris-HCl, pH 7.6, 50 mM MgCl2, 50 mM DTT,10 mM ATP)
Method
1 Prepare a 50 l reaction mixture containing 1.5 g blunt-end plasmid DNA, 1 ×Taq DNA polymerase buffer and 20 M dTTP.
2 Add 1 unit of Taq DNA polymerase and incubate at 70 °C for 1 h. This will promote the addition of a single dTTP residue as a 3’ extension to both termini of the plasmid DNA. This will allow subsequent cohesive ligation of the majority of PCR amplified DNA fragments which have a 3’ dATP extension.
3 Purify the plasmid DNA by phenol extraction and ethanol precipitation (see Purification of DNA by phenol extraction and ethanol precipitation) and resuspend the DNA at a concentration of 50 ng/l in sterile water.
4 Prepare a ligation mixture containing 0.25 units of T4 DNA ligase and an equimolar ratio of the plasmid DNA and PCR products (see DNA ligation). Incubate at 15 °C for 1–16 h.
5 The ligation is now ready for further analysis including transformation of E. coli cells.
