Alternative cloning method

Copyright information:Taken from "Gene C—Gene Cloning And Tagging for using yeast Homologous Recombination: a novel approach for the analysis of gene expression"Nucleic Acids Research 2005;33(18):e163-e163.Published online 27 Oct 2005PMCID:PMC1270953.© The Author 2005. Published by Oxford University Press. All rights reserved Two plasmids are required: () pC1 (SrfI-linearized) and () pC2 (HpaI-linearized). pC2 serves as a ‘stuffer fragment’ that contains the high-copy ColE1 origin of replication, exploited as described in , and a marker to prevent recircularization of empty vectors. () The first cloning step consists of generating a targeting vector. The yeast strain AB1380 is simultaneously transformed with four DNA fragments: SrfI-linearized pC1, HpaI-linearized pC2, and upstream and downstream linkers. Linkers are generated by PCR and contain 450–700 bp of homology to the target region boundaries flanked by 40 bp of sequence homologous to the pC1 and pC2 vector ends. The desired product from this transformation reaction results from four homologous recombination events, and consists of the stuffer fragment, flanked by target region boundaries, inserted in pC1. Transformation reactions are plated on selective media, and resulting plasmids are purified from yeast. () The next step involves gap repair of the targeting vector. The targeting vector is linearized with SrfI to release the ColE1 ori cassette, leaving behind free worm DNA ends with a overhanging fragment on the upstream end. A yeast strain bearing a YAC containing the desired worm target region is transformed with the SrfI-linearized targeting vector. The worm genomic region is gap repaired from the YAC into the targeting vector. () Correct plasmids contain the target region cloned into pC1. The remaining diagnostic and selection steps are identical to those described for the standard cloning method. See for legend details.