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Figure 2 | BMC Biotechnology

Figure 2

From: Single-cell duplex RT-LATE-PCR reveals Oct4 and XistRNA gradients in 8-cell embryos

Figure 2

Optimal DNA-polymerase concentration for amplification of Oct4 templates in single-amplicon vs duplex LATE-PCR assays. Fluorescent signals generated by Oct4 DNA under different amplification conditions. One-thousand mouse genome equivalents, containing 2000 copies of Oct4 DNA, were used for each assay; all tests were run in duplicates. For this experiment, the PCR profile described in the "Methods" section was slightly modified in order to highlight the Taq-polymerase concentration effect, shortening the second stage from 15 to 6 cycles, but similar results were obtained with either of the two PCR profiles. In one group of samples Oct4 was the only template amplified (shown by the thin lines). In a second group of samples Oct4 (shown by the thick lines) was co-amplified with Xist (not shown) by addition of Xist-specific primers. Increasing amounts of Taq polymerase were added to the samples in each group, as follows: red lines, 1 u per assay; blue lines, 2 u per assay; black lines, 3 u per assay. The presence of Taq at the highest concentration and of a second set of primers favors non-specific amplification with a negative effect on Oct4 amplicon production, as shown by the declining slope of the thick black lines. Thus, 2 u of Taq polymerase per assay represent the optimal enzyme concentration in the duplex, resulting in specific amplification with maximal efficiency (steepest slope).

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