Figure 2. Knock-out strategy and screening of mutant.

(A) Schematic overview of the knock-out strategy for shc. A neomycin (npt) cassette is inserted into shc by homologous recombination thereby deleting a part of shc. (B) PCR screening of genomic DNA for checking segregation of the mutant compared to wild type. Primers used for screening were a-d where a = shc_usR_as; b = shc_middle_F; c = shc_middle_R; d = shc_dsF_as. (C) RT-PCR of Δshc and wild type cDNA using reverse transcriptase (top) and without (bottom) as negative controls. Primers amplifying 23S were used as a control of equal loading of RNA.