Sensimix sybr no rox reagent

The abundances of ammonia-oxidizing archaea (using amoA gene), N 2 O reducing bacteria (using nosZ gene), and methanotrophs (using pmoA gene) were quantified on a CFX-96 thermocycler (Biorad, USA) using primers and conditions described in Table S1. Standard curves were generated using ten-fold serial dilutions of plasmids containing the correct insert of each respective gene. The 10 µl reaction mixture contained 5 µl SensiMix SYBR No-ROX reagent (Bioline, Sydney, Australia), 0.3 µl of each primer (20 mM), 0.4 µl BSA (20 mM), and 1 µl of diluted template DNA (1-10 ng). Melt curve analyses were conducted following each assay to verify the specificity of the amplification products, and the PCR efficiency for different assays ranged between 86-95%, 92-98%, and 96-99% for amoA, nosZ, and pmoA genes, respectively. Amplified products were run on a 2% agarose gel to confirm product size and specificity. Note that the AOB (ammonia-oxidizing bacteria) community was not included in our study because of low AOB abundance according to our results from qPCR.
Thus, the PCR products did not satisfy the requirements for T-RFLP (next section). The same problem has been reported in a previous study including samples from a region near our sampling locations (Liu et al., 2016) .

The Bio-Rad CFX384 TM Real-Time PCR Detection System (Bio-Rad, Herculers, CA, USA) was used to qualify the copA and pcoD genes using the primer pairs copAF2010/copAR2333 (Li et al. 2012 ) and pcoR1/pcoR2 (Trajanovska et al. 1997) , respectively. The 10 µl PCR reaction mixture consisted of 5 µl SensiMix SYBR No-ROX reagent (Bioline), 0.5 µl of each primer (10 µM), 2 µl of 10-fold diluted DNA template, and 2 µl of nuclease-free water. The thermal-cycling conditions were: 95C for 10 min, 40 cycles of 95C for 30 s, 51C for 30s, and 72C for 45 s. The bacterial 16S rRNA gene was quantified using the primer pair BACT1369F/PROK1492R (Suzuki et al. 2000) . Amplification conditions were: 95C for 10 s, 35 cycles of 95C for 15 s and 56C for 1 min. The CRGs (i.e.
the copA and pcoD genes) and the bacterial 16S rRNA gene were PCR-amplified from the soil DNA, linked to the pGEM ® -T Easy Vectors, and transferred to JM109 High Efficiency Competent Cells (Promega, Madison, USA). Plasmid DNA was extracted from positive clones using the PureYield TM Plasmid Miniprep System (Promega) and sequenced to confirm their identities. Standard curves were prepared from 10-fold serial dilutions of plasmids with inserts of the targeted gene sequences. We conducted melting curve analysis after each qPCR run to verify the specificity of PCR amplicons.