A Simple Method for Constructing Cloning Vehicles with Long Polylinker Sequences (Commemoration Issue Dedicated to Professor Mituru Takanami On the Occasion of His Retirement)
Nobuo Nomura, Satoshi Tabata · Kyoto University Research Information Repository (Kyoto University) · 1993
A rapid method for constructing new cloning vectors was described.The special features taken for mutagenesis and selection are as follows : (i) mutagenesis was carried out by hybridizing oligonucleotides to single-stranded (SS) template DNA ; (ii) oligonucleotides were designed to harbor short stretches complementary to SS DNA at their both ends ; (iii) SS DNA for template was prepared from the BW313 strain ( dut-ung-) and transformation following hybridization and repair was carried out in JM109 strain (dut+ung+) in order to enrich an objective construct; (iv) blue-colored colonies (plaques) were selected in the X-gal plates among white-colored colonies (plaques) which are the starting clones and the integrity of the polylinker region was confirmed by sequencing.We describe the construction of new cloning vehicles derived from pUC118 and mp18, and discuss general application of this method to construction of new cloning vectors.