Icycler iq5 multicolor

Total RNA was extracted from harvested GCO cells using Trizol (Accurate Biology Co. Ltd., Shenzhen, China) according to the manufacturer’s protocol. The quality and the purity of isolated RNA were determined using a Nanodrop 2000c spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA was reverse-transcribed to cDNA using the HiScript Q Select RT Supermix kit (Vazyme, Nanjing, China) for qPCR (+gDNA wiper). The qRT-PCR protocol was as follows: 95 °C for 4 min, and then 38 cycles at 95 °C denaturation for 10 s, followed by annealing at 59 °C for 30 s with a Bio-Rad iCycler IQ5 Multicolor real time PCR detection system. Table 1 shows the primers used for qRT-PCR in the study.

Copy number of 16S rDNA and nifH genes from collected soil samples were quantified using iCycler iQ5 Multicolor (Bio-Rad Lab, Hercules, USA) real-time polymerase chain reaction (qPCR) machine as described previously [17 , 20 ]. DGGE was performed on a Dcode system (Bio-Rad Lab, Hercules, CA, USA). PCR for 16S rDNA and nifH DGGE analysis was performed as per earlier studies and PCR products were separated on 8% (w/v) acrylamide–bisacrylamide gel with a 40–70% denaturing gradient of urea and formamide at 60°C and 90V in 1X TAE for 16 hours [20 , 21 ]. The gels were stained for 30 min in ethidium bromide in 1X TAE (Invitrogen, Paisley, UK) and visualized with a Gel Documentation system (Bio-Rad Lab, Hercules, CA, USA).