Axyprep bacterial genomic dna miniprep kit

The 16S rRNA gene was amplified and analyzed as described previously [13 (link)]. PCR products were cloned into the vector pMD 19-T (TaKaRa, Dalian, China) and then sequenced to determine the almost-complete sequence of the 16S rRNA gene. High-quality genomic DNA was extracted with the AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Axygen Scientific, Inc., Union City, CA, USA). The genomes of strains JW1^T, JW3, and P. byunsanensis JCM 12483^T were sequenced using the Solexa paired-end sequencing technology with the Illumina HiSeq 2000 platform (Anoroad Gene Technology Co. Ltd, Beijing, China). One paired-end library was constructed with 500-bp insert size. The sequencing generated approx. 1 Gb of clean data (approx. 500-fold genome coverage). De novo assembly of the reads was carried out using SOAPdenovo (version 2.0.1) [14 (link)]. Assembly k-mer was tested from 57 to 64 for seeking the optimal value, using the abyss-pe script. Assembly quality was estimated using MUMmer [15 (link)]. Completeness of the genome sequence was addressed using the bioinformatics tool CheckM (http://ecogenomics.github.io/CheckM/) [16 (link)]. The complete sequence of the 16S rRNA gene was annotated via the RNAmmer 1.2 Server [17 (link)] and was compared with related sequences of reference organisms using the EzTaxon-e service [18 (link)].

Genomic DNA from strain RZC12 was extracted with an AxyPrep Bacterial Genomic DNA Miniprep Kit (Axygen Scientific, USA) in accordance with the manufacturer’s instructions. The PCR amplification of 16S ribosomal RNA was performed using primers fD1: (5′-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3′) and rD1: (5′-CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3′) [27 (link)]. The program used for PCR amplification included the following steps: initial denaturation at 94 °C for 3 min, followed by 30 cycles at 94 °C for 1 min, annealing at 55 °C for 45 s, and extension at 72 °C for 1.5 min, and then final extension at 72 °C for 5 min using an Eppendorf thermocycler (Eppendorf AG, Hamburg, Germany). The amplified fragments were purified with the AxyPrep PCR Cleanup Kit (Corning Life Sciences, Tewksbury, MA, USA) in accordance with the manufacturer’s instructions and subsequently sequenced by a commercial service (Macrogen Inc., Seoul, Korea). The strain sequence was identified through a search of the NCBI database [28 ]. Related sequences were retrieved from the Genbank database, and a multiple alignment was generated using Clustal X2 [29 (link)]. A phylogenetic analysis was performed using the neighbor-joining method using the software MEGA 11 [30 (link)].

Genomic DNA was extracted using the AxyPrep bacterial genomic DNA miniprep kit (Axygen Biosciences, Union City, CA, USA) from freshly overnight cultures following the manufacturer’s instructions. PCR was carried out on a thermal cycler system T100 (Bio Rad, Hercules, CA, USA) using PCR Master Mix (Fermentas Life Sciences, Vilnius, Lithuania), The PCR amplifications were examined by electrophoresis purification was done using a QIA quick PCR purification kit (Qiagen, Hilden, Germany). The primers used are as listed in (Table 1) and obtained from Bioserve Company (Cairo, Egypt).