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Product identities generated by each primer set were subsequently confirmed by sequencing.
Product identities were confirmed by agarose gel electrophoresis and PCR product sequencing (Robarts Research Institute sequencing facility, London, Ontario).
The product identities were unequivocally confirmed by NMR spectroscopy and exhibited nearly identical diagnostic C chemical shifts of ∼147 and ∼133 ppm and a J HH coupling constant of ∼16 Hz.
PCR product identities were initially confirmed as either UGT1A1 or UGT1A6 (and not any other UGT1A gene) by phylogenetic tree analysis (neighbor-joining) inputting all available mammalian UGT sequences (listed at http://www.flinders.edu.au/medicine/sites/clinical-pharmacology/ugt-homepage.cfm).cfm
Throughout real-time PCR analysis, product identities were confirmed by melting curve analysis.
Further confirmation of the product identities was obtained by immunoblot probing for hGH, MBP, and hIgG.
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