title: Anaerobic sludge
design: The hypervariable region V4 of the 16S rRNA gene were amplified with primer pairs 515FmodF (5'-GTGYCAGCMGCCGCGGTAA-3') and 806RmodR (5'-GGACTACNVGGGTWTCTAAT-3') (Sampson et al., 2016) by T100 Thermal Cycler PCR thermocycler (BIO-RAD, USA). The PCR reaction mixture consisted of 10 L of 2  Pro Taq, 0.8 L of 5 M Forward Primer, 0.8 L of 5 M Reverse Primer, 10 ng/L of template DNA, and ddHO to reach a final volume of 20 L. The cycling conditions for PCR amplification were as follows: First, an initial denaturation step was carried out at 95 for 3 minutes. Then, 27 cycles were performed, each cycle including denaturation at 95 for 30 seconds, annealing at 55 for 30 seconds, and extension at 72 for 45 seconds. After that, a single extension step was conducted at 72 for 10 minutes, and the reaction ends at 10. The PCR product was extracted from a 2% agarose gel. Subsequently, it was purified using the PCR Clean-Up Kit (YuHua, Shanghai, China) in accordance with the manufacturer's instructions. Finally, the product was quantified using Qubit 4.0 (Thermo Fisher Scientific, USA).
identifiers.namespace: SUB15371443
identifiers.id: RB_20240818