Picogreen dsdna quantitation assay

About 1.5 g of the surface-sterilized plant sample was frozen with liquid nitrogen and ground to a fine powder in a sterilized and precooled mortar. DNA was extracted using a Plant DNA Kit (Omega Bio-Tek, Norcross, USA) and the V3-V4 hypervariable region of the bacterial 16S rRNA gene amplified using Bac 341f (5′-CCTACACGACGCTCTTCCGATCTN-3′) and Univ 805r (5′-GACTGGAGT TCCTTGGCACCCGAGAATTCCA-3′) primers. The 50 μl PCR reaction mixture contained 100 ng of DNA extract, 1× Taq reaction buffer, 20 pmol of each primer, 200 μM each dNTP and 1.5 U of Taq DNA polymerase (Sangong Biotech, China). DNA aliquots were PCR-amplified with 5 min denaturation at 95 °C, 30 cycles of 1 min at 94 °C, 50 s at 55 °C, and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. All samples were amplified. Replicate PCR products of the same sample were assembled within a PCR tube. Then they were visualized on agarose gels (2% in TBE buffer) containing ethidium bromide, and purified with a DNA gel extraction kit (Sangong Biotech, China). After purification, the concentration of the PCR products was measured on Qubit^®2.0 Fluorometer using the PicoGreen^® dsDNA quantitation assay (Invitrogen, Carlsbad, CA). DNA concentration was adjusted to 1 ng/ml. The amplicon libraries were prepared by pooling 10 ng of each PCR.