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Recombineering Homologous Recombination Constructs in Drosophila
Published on: July 13, 2013
A method for filling in the cohesive ends of double-stranded DNA using Pfu DNA polymerase
Shaohui Yang1, Xin Li, Dongfeng Ding
1The Key Laboratory of Bioactive Materials, Ministry of Education/Institute for Molecular Biology, Nankai University, Tianjin 300071, People's Republic of China.
Abstract:
The present paper reports a highly efficient method of making blunt ends from cohesive ends of double-stranded DNA. Klenow fragment and Pfu DNA polymerases were used to fill in the cohesive ends. Since the transformation efficiency can directly reflect the filling-in efficiency, similar ligation and transformation conditions were used, and the filling-in efficiency was compared with the corresponding transformation efficiency. The results indicate that the filling-in efficiency of Pfu DNA polymerase was 1.96 times that of Klenow fragment and its efficiency was markedly higher than that of Klenow fragment (P<0.01). The optimization experiments on reaction conditions indicate, when the pH is 8.5 and the temperature is 74 degrees C, that the filling-in efficiency was highest upon using a buffer containing 3 mM MgSO4 and 300 microM dNTP.
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