Subcloning of large DNA regions from BACs. (A) BAC subregions were subcloned into a common acceptor vector using targeting 80-bp linkers (inset). The common vector, pCRG16, is a yeast ↔E. coli shuttle plasmid that carries the yeastCEN6/ARSH4 segregation/replication functions and the yeastURA3 selectable marker. For replication and selection inE. coli, the plasmid contains the single-copy mini F‘ replication/segregation functions and the chloramphenicol-resistance marker, respectively. For BAC subcloning, 100 ng ofSrfI-digested pCRG16 acceptor vector, 1 μg (20 pmole) of each linker, and 1 μg BAC DNA digested with AscI orNotI and combined in equal amounts were cotransformed into yeast spheroplasts. (B) Transformation plates of spheroplasts transformed with vector alone or vector + linkers + BAC DNA and plated on Ura-deficient media or Ura-deficient media containing 2.5 μg/mL of cycloheximide. The RPCI-11–98D12L cotransformation is shown. (C) Schematic of left and right target-insert PCR assay strategy and whole-yeast-cell PCR data for eight representative colonies from the RPCI-11–98D12L subcloning. Double-positive clones are shown highlighted in boldface type. (D) Yield of PCR double-positive yeast colonies from three representative BAC subcloning experiments.
