T/A-based subcloning of PCR amplified DNA fragments

2010-03-05 11:59 · rose

Richard Powell Department of Microbiology, National University of Ireland, Galway, Ireland Frank Gannon European Molecular Biology Labora

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.

 

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