Geneamp pcr system

RNA preparation and reverse transcription were performed as previously described.¹⁸ The RT‐PCR exponential phase was set for 30 cycles to allow for quantitative comparison of the various cDNAs developed from identical reactions on a GeneAmp PCR system (Eppendorf). Real‐time PCR was carried out using a Rotor‐gene (Qiagen). Reactions were run in triplicate in three independent experiments. Expression data were normalized to the geometric mean of the housekeeping gene GAPDH to control for variability in expression levels and were analysed as previously described.²³

Molecular identification of the most NiRB was performed by 16S rDNA PCR Sequencing [16 (link)]. Genomic DNA was extracted and the 16S rDNA gene was amplified by using the universal bacterial Primers of F27 (5′-CAGGGTACCAGAGTTTGA-3′) andR1492 (5′-CTCTCTGCAGTACGGCTAC-3′).
PCR was performed as a 25 μL reaction Mixture containing 2 μL of DNA extract as a template. Each primer at a concentration of 8 PM, 2 mM mgCl₂ and dNTP_S at a concentration of 0.2 mM, as well as 1.25 U of Taq polymerase and buffer were used. After the initial denaturation for 5 min at 95°C, there was 32 Cycle consisting of denaturation at 94°C for 1 min, annealing at 57°C for 1 min, extension at 72°C for 1 min and final extension at 72°C for 5 min. PCR was carried out in a gene AMP PCR system (Eppendorf). PCR products were analyzed by 1% (W/V) agarose gel electrophoresis in 0.5x TBE buffer with ethidium bromide (05 μ_g/ml). PCR products were then sequenced and compared with the National Center for Biotechnology Information (NCBI) database using the BLAST search available through the center’s website (http://www.ncbi.nlm.nih.gov/BLAST). The 16S rDNA sequences were then submitted to the Gene Bank using the BankIt service.