Absolute blue qpcr sybr green master mix kit

All qRT-PCR reactions were performed in Eppendorf Mastercycler^®ep realplex thermal cycler using the intercalation dye ABsolute Blue QPCR SYBR Green master mix kit (Thermo Scientific) as a fluorescent reporter. All PCR reactions were performed in triplicates for three biological replicates in 25 μl volumes using 1 μl of each forward and reverse primers (25 pmol each), 12.5 μl of SYBR green master mix, 1 μl of cDNA (100 ng/μl), and 9.5 μl HPLC molecular biology grade water. RNAs and cDNAs were prepared from Camelina seeds harvested between 10 and 16 days after flowering (DAF), and PCR products were quantified, using specific PCR primers for the gene of interest, in the qPCR cycling program of 1 cycle at 95 °C for 15 min, 30–40 cycles at 95 °C for 15 s, 50–60 °C for 30 s, and 72 °C for 30 s. The quantification of PCR products was performed using the 2^−ΔΔCt method [83 (link)], and the Camelina β-actin gene was used as internal reference to normalize the relative amount of mRNAs for all samples. The error bars represent the standard errors for the fold changes of relative gene expression calculated from at least two independent biological replicates and triplicate PCR reactions for each sample. A list of PCR primers used is presented in Additional file 1: Table S16.

All qRT-PCR reactions were performed in Eppendorf Mastercycler^®ep realplex thermal cycler using the intercalation dye ABsolute Blue QPCR SYBR Green master mix kit (Thermo Scientific) as a fluorescent reporter. All PCR reactions were performed in triplicates for three biological replicates in 25 µl volumes using 1 µl of each forward and reverse primers (25 pmol each), 12.5 µl of SYBR green master mix, 1 µl of cDNA (100 ng/µl), and 9.5 µl HPLC molecular biology grade water. The cDNAs were amplified, and PCR products were quantified, using gene-specific primers, in the qPCR cycling program of 1 cycle at 95 °C for 15 min, 30–40 cycles at 95 °C for 15 s, 50–60 °C for 30 s, and 72 °C for 30 s. The quantification of PCR products was performed using the 2-ΔΔCt method [40 (link)], and the Camelina β-actin gene was used as internal reference to normalize the relative amount of mRNAs for all samples. The error bars represent the standard errors for the fold changes of relative gene expression calculated from two independent biological replicates and triplicate PCR reactions for each sample. Flowers and leaf samples were used here as controls. The PCR primers for candidate genes quantified by qRT-PCR are summarized in Additional file 1: Table S1.