Rescuing YAC-insert ends as E. coli plasmids.
End clones from YACs (terminal fragments of the YAC-insert DNA cloned into a plasmid vector) are essential ingredients for contig building using chromosome walking strategies, for fluorescent in situ hybridization experiments, and for generating repeat-free probes for pulsed-field gel electrophoresis or genetic linkage analysis. The basic protocol describes a method for rescuing the CEN (centromere) ends of YACs constructed in the vector pYAC4. However, because this method relies on XhoI and SalI sites, which are relatively rare in the mammalian genome, it is not always possible to obtain a subclone in this manner. An alternate protocol presents a method utilizing integrative plasmid-rescue vectors to facilitate the isolation of both ends of any YAC clone even in the absence of convenient restriction enzyme sites.
Hermanson
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Cytel Corporation, San Diego, California, USA.
Evans
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D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors; D005826:Genetics, Medical; D006801:Humans; D010957:Plasmids / Q000235:genetics*