2.4.1 Soil bacterial DNA extraction and PCR amplification
The total DNA of soil samples was extracted using the Power soil DNA
Isolation Kit (MOBIO, USA), with 9 DNA samples for 9 mixed soil samples.
Nanodrop 2000c was used to detect the quality of DNA, followed by PCR
amplification of the bacterial 16S rRNA gene. The amplification primers
were 338F (ACTCCTACGGGAGGCAGC AG) and 806R (GACTACHVGGGTWTCTAAT). The
PCR conditions were as follows: predenaturation at 98°C for 3 min;
denaturation for 30 s at 98°C; annealing at 50°C for 30 s; 72°C
extension for 30 s; 27 cycles; maintenance at 72°C for 5 min; and
storage at 4°C. The electrophoretic PCR products were gelled and
purified using an AxyPrep DNA gel recovery kit (AXYGEN). The recovered
and purified PCR products were then quantified using the
QuantiFluorTM-ST blue fluorescence quantification system (Promega, USA).
According to the quantitative results and sequencing quantity
requirements, PCR products were used to construct a sequencing library.
The constructed library was sequenced on the IlluminaPE300 sequencing
platform.