In vivo or gap-repair cloning in yeast has been widely recognized as one of the most efficient means for error-free construction of plasmids. A protocol is described here that allows easy and efficient gap-repair cloning that is based on two major modifications. Instead of subcloning, the targeting plasmids are constructed using oligonucleotides from sequences derived from the upstream and downstream sequences of the fragment to be cloned. These sequences are selected so that they can lead to the generation of recognition sites for restriction enzymes that produce blunt ends. Accordingly, this procedure can be applied to any DNA fragment, regardless of whether these include unique restriction sites to generate the targeting ends. With the strategy described, ≃50 bp upstream and downstream targeting ends are generated that allow efficient cloning. Further, to allow easy identification of the positive clones, the annealed oligonucleotides are cloned in frame with the lacZ fragment present in the plasmid. Accordingly, these plasmids produce blue Escherichia coli colonies on media containing X-Gal. On the other hand, plasmids rescued from yeast that have acquired the respective cognate sequences produce white colonies. To demonstrate the efficiency of the method, this report includes the cloning of fragments harbouring the CDC28, CAK1, CIN5 and CLB2 genes. We found that 30-100% of the analysed plasmids carried the expected inserts. Copyright © 2009 John Wiley & Sons, Ltd.