Schematic Outline of the Cosmid Complementation Strategy
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posted on 2008-02-15, 02:14 authored by Marc-Jan Gubbels, Margaret Lehmann, Mani Muthalagi, Maria E Jerome, Carrie F Brooks, Tomasz Szatanek, Jayme Flynn, Ben Parrot, Josh Radke, Boris Striepen, Michael W WhiteConfirmed mutants are transfected with the ToxoSuperCos cosmid library (containing 40–50 kb inserts of WT RH genomic DNA), and selected under pyrimethamine at the restrictive temperature. Sequence tags of complementing cosmids are rescued into plasmid from parasite genomic DNA by restriction, dilute ligation and selection for kanamycin resistance after electroporation into E. coli (insert can be sequenced using T3 primer). Querying ToxoDB using independent sequence tags (shown in blue) identifies a minimal complementing locus (dashed vertical lines). The locus is confirmed and narrowed using a library of arrayed and sequenced cosmids (shown in red) and/or PCR products (shown in black) covering individual genes.
