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. 2009 Aug 27;4(8):e6805. doi: 10.1371/journal.pone.0006805

Figure 3. Southern blot and PCR analysis of GLOI knockout mutants.

Figure 3

Southern blot analysis of wild type and mutant parasites to confirm the recombination events. Equal amount of genomic DNA (5 µg) was digested with SalI restriction enzyme and separated on a 0.8% agarose gel. The DNA was blotted on to membrane and hybridized with hyg specific probe (A), NEO specific probe (B), GLOI specific probe (C) and PHLEO specific probe (D). (+/+) represents Wild type; (+/h) represents heterozygote LdGLOI::HYG clone; (+/n) represents heterozygote LdGLOI :: NEO clone; (+/h/n) represents L. donovani double targeted transfectant, LdGLOI :: HYG :: NEO clone, (+/h-GLOI+) represents GLOI complementation mutant and (−/− GLOI+) represents GLOI null mutants rescued with an episomal copy of GLOI. M represents DNA molecular weight marker; uncut pX63-HYG and pX63-NEO and linearized pGEM7zfαNeoα-GLOI+ plasmids were used as positive controls for the respective blots probed with HYG, NEO and GLOI. PCR was carried out with primers P1 and P2 for checking hyg cassette integration (E, panel a) and with primers P1 and P3 for checking neo cassette integration (F, panel a). Southern blot analysis of the corresponding PCR gels probed with HYG (E, panel b) and NEO (F, panel b). G: Southern blot analysis of genomic DNA digested with SalI and probed with PHLEO probe to verify phleo cassette integration.