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Sequence of the synenvBF was corroborated by nucleotide sequencing of the DNA extracted from the 3 kpb band product obtained after the "integrity PCR".
The PCR-amplified DNA fragments of all samples showed a clean single band product when examined on an agarose gel (Figure 3).
The lower band (product a) contains one phosphate group (×1), while the upper (product b) band contains two or three phosphate groups (×2.5).
Each reaction involving the synthesis of polA resulted in a single band product.
All primer sets were confirmed to produce a single band product using conventional gel-PCR and a single peak in melting curve analysis, which was performed for every well at the conclusion of the experiment.
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A high annealing temperature was used so that single band products, if present, could be later sequenced.
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All PCR primer pairs (Additional file 5: Table S4) were optimized to produce single band products when run on the Agilent Bioanalzyer 2100.
To confirm specific amplification of the primers and probes, randomly selected PCR products were run on an agarose gel, revealing single band products that when sequenced corresponded to the region of interest (Loyo et al, 2010).
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