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Earthworms were dissected to obtain the gut contents. Then, 660 μL PB solution (112.87 mM Na2HPO4, 7.12 mM NaH2PO4) and 220 μL TNS solution (0.5 M Tris HCl at pH 8.0, 0.1 M NaCl, 10% SDS) were added to a 2-mL centrifuge tube with a 0.2 g sample of earthworm gut content. After 50 s vibration (6.5 m/s), the tube was centrifuged for 5 min at 14,000 rpm and 4ºC. Then, 750 μL liquid supernatant was transferred to a new centrifuge tube and extracted in 750 μL PCI solution (the ratio of phenol, chloroform and isoamyl alcohol was 25:25:1) and precipitated in isopropanol. The V4 region of the bacterial 16S rDNA gene was amplified to construct the sequencing library. |
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PCR |
PAIRED
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