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
Klenow DNA polymerase
10× Klenow reaction buffer (500 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM DTT)
Stock solutions (e.g. 20 mM) of each dNTP (i.e. dATP/dCTP/dGTP/dTTP)
T4 DNA polymerase
10× T4 pol reaction buffer (500 mM Tris-HCl, pH 8.8, 50 mM MgCl2, 50 mM DTT)
Water-bath or heating-block
Gel electrophoresis apparatus
A. Filling in 5'-overhangs with Klenow DNA polymerase
1 Prepare a reaction mixture containing DNA, 1 × Klenow reaction buffer and 20 M of each dNTP.
2 Add 1 unit of Klenow DNA polymerase and incubate at 15–37 °C for 15–30 min.
3 Stop the reaction by heating at 75 °C for 10 min.
4 The efficacy of the reaction can be monitored by a simple ligation experiment followed by gel electrophoresis. DNA that originally had complementary termini should not show no ligation ability when using low amounts of T4 ligase suitable only for cohesive-end ligation (see DNA ligation). An approximate ten-fold increase in the amount of ligase should now be required to achieve the necessary blunt-end ligation.
B. Removal of 3'-overhangs by the exonuclease reaction of T4
DNA polymerase
1 Prepare a reaction mixture containing DNA, 1 × T4 DNA pol reaction buffer and 20 M of each dNTP.
2 Add 1 unit of T4 DNA polymerase and incubate at 15 °C for 15–30 min.
3 Stop the reaction by heating at 75 °C for 10 min.
4 Analyse the efficacy of the reaction by ligation of a sample as described in step A.4 above.
