Hard shell white pcr plates

cDNA synthesis was performed using SuperScript II reverse transcriptase and random primers (Invitrogen) in the presence of RNasin ribonuclease inhibitor (Promega). Hundred nanogram of total RNA was used. cDNA was then used as template in qRT-PCR performed with a SensiFAST SYBR No-ROX kit (Bioline). Three liquid culture biological replicates and three rhizosphere biological replicates were used for each gene. Specific qPCR primers (numbers 1–14) were used to amplify reference and target genes. To normalize for differing primer efficiency, a standard curve was constructed (in duplicate) using chromosomal DNA. Melting curve analysis was used to confirm the production of a specific single product from each primer pair. qRT-PCR was performed using a CFX96 Touch instrument using hard-shell white PCR plates (Bio Rad). PCR products were detected with SYBR green fluorescent dye and amplified according to the following protocol: 95°C, 3 min, then 50 cycles at 95°C 5 s, 62°C 10 s and 72°C 7 s. Melting curves were generated at 65–95°C with 0.5°C increments. Primers were used at a final concentration of 250 nM.

cDNA synthesis was performed using SuperScript II reverse transcriptase and random primers (Invitrogen) in the presence of RNasin ribonuclease inhibitor (Promega). The quantity of total RNA used was dictated by the lowest concentration sample in each assay in the case of rhizosphere samples. cDNA was then used as template in qRT-PCR performed with a SensiFAST SYBR No-ROX kit (Bioline). Three technical replicates were used for each gene. Specific qPCR primers (31–34 and 70–73) were used to amplify reference and target genes. To normalize for differing primer efficiency, a standard curve was constructed (in duplicate) using chromosomal DNA. Melting curve analysis was used to confirm the production of a specific single product from each primer pair. qRT-PCR was performed using a CFX96 Touch instrument using hard-shell white PCR plates (Bio Rad). PCR products were detected with SYBR green fluorescent dye and amplified according to the following protocol: 95°C, 3 min, then 50 cycles at 95°C 5 sec, 62°C 10 sec and 72°C 7 sec. Melting curves were generated at 65 to 95°C with 0.5°C increments. Primers were used at a final concentration of 1 μM. The entire experiment (including RNA extraction) was repeated once independently.