Maxima sybr green 2 pcr mastermix

Trace amounts of contaminating DNA were removed from RNA samples (1 μg) by treatment with RNase-free DNase I per the manufacturer’s protocol (Thermo Fisher). RNA was reverse transcribed using the RevertAid first-strand kit and primed with random hexamers per the manufacturer’s protocol (Thermo Fisher). cDNA (100 ng) was amplified using gene-specific primers (Sigma) and the Maxima Sybr green 2× PCR mastermix per the manufacturer’s protocol (Thermo Fisher). Amplification and fluorescence measurement were conducted using the 7500 real-time PCR system platform (Applied Biosystems). Expression levels of target genes in polymicrobial cultures were compared to those in monomicrobial cultures and normalized to the staphylococcal reference gene gyrB using the ΔΔC_T method as described previously (67 (link)). RNA was extracted from triplicate experiments, and ΔΔC_T values were calculated, averaged, and expressed as the mean ± SD.

Total RNA isolation was carried out from all 5 samples, one control and 4 stress samples using TRIzol method and cDNA synthesis Kit (ThermoScientific, USA) was used for synthesis of template cDNAs from 2 µg of total RNAs. qRT-PCR was performed following the SYBR Green chemistry (Maxima SYBR Green 2 × PCR Master Mix, ThermoScientific, Waltham MA, US) and Fast Real Time PCR system (7900HT Applied Biosystems, USA) for validation of the Illumina sequencing data following the protocol described by Rastogi et al.^{19 (link)}. Relative mRNA levels were quantified with respect to the endogenous control gene ‘actin’ of A. annua. All the experiments were repeated using three biological replicates and statistical analysis (±Standard Deviation) of data was carried out. For PCR amplification of stress specific transcripts, primers were designed from the transcriptome sequences and cDNAs synthesized above were used as template. Amplification conditions were as: 95 °C for 1 min followed by 35 cycles of 95 °C for 30 sec, annealing temperature ranging from 48 °C–55 °C for 30 sec, 72 °C for 1 min 30 sec, followed by a final extension of 5 mins at 72 °C. 1.2% agarose gel was prepared to electrophorese and visulalize the amplified product.