For real-time PCR, gene-specific oligonucleotide primers were designed and are described in Supplementary Table S7 at JXB online. The gene specificity of each pair of primers was double checked by melting curves and product resequencing, according to the procedures described by Yin et al. (2008) (link). The EjACT gene was employed as the internal control for monitoring the abundance of the mRNA. The sequences of EjACT primers are described in Supplementary Table S7 .
Real-time PCR were performed on a LightCycler 1.5 instrument (Roche), initiated by 5 min at 95 °C and followed by 45 cycles of 95 °C for 5 s, 60 °C for 5 s, and 72 °C for 10 s, and completed with a melting- curve analysis program. The PCR mixture (10 μl total volume) comprised 2 μl of 5× LightCycler FastStart DNA MasterPLUS SYBR Green I Master Mix (Roche), 0.5 μl of each primer (10 μM), 1 μl of diluted cDNA and 6μl PCR-grade H2O. No-template controls and melting-curve analysis were included for each gene during each run.
Real-time PCR were performed on a LightCycler 1.5 instrument (Roche), initiated by 5 min at 95 °C and followed by 45 cycles of 95 °C for 5 s, 60 °C for 5 s, and 72 °C for 10 s, and completed with a melting- curve analysis program. The PCR mixture (10 μl total volume) comprised 2 μl of 5× LightCycler FastStart DNA MasterPLUS SYBR Green I Master Mix (Roche), 0.5 μl of each primer (10 μM), 1 μl of diluted cDNA and 6μl PCR-grade H2O. No-template controls and melting-curve analysis were included for each gene during each run.
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