16S rRNA gene sequences amplified from grassland soil at the total (DNA) level: CT2B
The V4 region of the 16S rRNA gene in total soil DNA and cDNA samples were amplified using bacterial and archaeal primers 515F and 806R (Caporaso et al., 2011, 2012) as recommended by the Earth Microbiome Project. Amplification of 16S rRNA gene from DNA and cDNA samples were carried out in 50-無 reaction volumes containing final concentrations of: 5 ng/無 of genomic DNA or 1 無 of cDNA as template, 1_ KAPA HiFi Fidelity Buffer with 2.0 mM MgCl2 (Kapa Biosystems), 300 然 of each dNTP, 300 然 of each primer and 1 U KAPA HiFi DNA Polymerase (Kapa Biosystems). The reactions were held at 95。 for 3 min, followed by 25 cycles of 98。 for 20 s, 60。 for 30 s, 72。 for 30 s and a final extension of 72。 for 1 min. Second round of PCR was performed with 2 ng of purified amplicons (from first round amplification) as template using the same reaction concentrations as the first round. The second round PCR conditions were 95。 for 20 s, followed by 10 cycles of 98。 for 20 s, 60。 for 20 s, 72。 for 20 s and a final extension of 72。 for 1 min.