Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.