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In addition, the present cassette analysis allowed the simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples.
The sequences obtained from the gene cassette analysis have been deposited in GenBank/EMBL/DDBJ under accession numbers JX195555 for aadA6-ofrD of P. aeruginosa strain ODM-24 and JX195556 for aadA13 of strain ODM-08.
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We expect that this approach will be useful for the construction of strains carrying multiple expression cassettes for analysis of trypanosomatid pathogenicity mechanisms and overexpression of multi-subunit protein complexes for biochemical and structural studies.
Samples were then dispensed immediately onto commercially available test cassettes for analysis in a Cholestech LDX System (Alere Inc., Waltham, MA) according to strict standardized operating procedures.
PC12 20.4 cells [16] contain a stably integrated CMV-LoxP-STOP-LoxP-EGFP cassette which enables analysis of Cre mediated DNA recombination by EGFP fluorescence.
Offspring were screened for removal of the neomycin cassette by PCR analysis.
To further verify the precise quantitative patterns of exon inclusion of the genes where the end-point quantification was not conclusive, we selected five of the already confirmed alternative cassette exons for analysis using quantitative real-time PCR.
However, to rigorously demonstrate that only the cell inheriting the Watson strand is competent for switching, integration of an extra inverted cassette with subsequent analysis of broken molecules and their switching mode is required, just as what was performed decades ago in experiments with S. pombe (Klar 1987b , 1990.
These clones were also checked for the correct integration of the phleo replacement cassette by Southern blot analysis of SalI digested genomic DNA followed by hybridization with the phleo probe.
We observed its impact in our analysis of cassette exon pairs.
However, most studies were restricted to the analysis of cassette exons in pairs of genomes and did not analyze functionality of the alternative variants.
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