Rapid bacterial genomic dna isolation kit

Sphingopyxis sp. USTB-05 was initially incubated on the original solid isolation media at 30 °C for 48 h. A single colony was selected and cultivated in the culture medium of previous report [22 (link)]. The genomic DNA was extracted using the Rapid Bacterial Genomic DNA Isolation Kit (CoWin Biosciences, Taizhou, Jiangsu, China) according to the manufacturer’s instructions. NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) analysis and gel electrophoresis were used to determine the purity and concentration of the DNA samples. A small fragment second-generation genomic library with a size of 350 bp was constructed using the NEBNext^® Ultra™ II DNA kit. The genome was sequenced by using Illumina X10 platform (Madison, WI, USA) [47 (link)]. The third-generation genomic library was structured by the standard protocol of Oxford Nanopore Technologies (ONT, Oxford, UK).

The colonies were obtained after 48 h of incubation on LB agar medium at 30 °C. A single colony was picked and cultured for 48 h at 30 °C, 200 rpm to logarithmic phase in LB liquid media. Cells were collected by centrifugation at 13,282× g for 10 min at 4 °C, and the supernatant was discarded. Genomic DNA was extracted using the Rapid Bacterial Genomic DNA Isolation Kit (CoWin Biosciences, Taizhou, Jiangsu, China) according to the manufacturer’s instructions. DNA quality, concentration and integrity were checked by NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) and gel electrophoresis. DNA damage repair and end repair, magnetic beads purification and linker connection were processed by using the official SQK-LSK ligation kit (BAIYITECH, Hangzhou, Zhejiang, China). The third-generation genomic library was constructed by the standard protocol of Oxford Nanopore Technologies (ONT, Oxford, UK). High quality data sets with corresponding sequencing depths of 100-fold were generated (Figure S1). Large fragments of DNA were recovered by using BluePippin (Notre Dame, IN, USA) automatic nucleic acid recovery system [16 (link)]. The second-generation genomic library of strain NBD5 was performed using the Illumina X10 platform (Madison, WI, USA) [17 (link)].