Pdrive pcr cloning vector

A phylogenetic characterisation based on molecular taxonomy was performed by sequence analysis of the 16S rRNA gene. Genomic DNA of all strains was isolated using the Qiagen DNeasy Blood and Tissue Kit. The 16S rRNA gene was amplified by PCR using the universal bacterial 16S rDNA primers F27 and R1492 [16] . Obtained PCR products were cloned into the Qiagen pDrive PCR cloning vector and sequenced using standard M13 vector primers at MWG Biotech (Germany). DNA sequences of partial 16S rRNA genes (average length 1470 bp) were compared to those available in the GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html) using nucleotide BLAST [17] (link). Sequences were aligned and a phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 4 [18] (link). The tree was computed using the neighbor-joining method and the resulting tree topology was tested by bootstrap analysis performed with 1000 replicates. The 16S rRNA gene sequence of Bacillus subtilis (GenBank accession number AJ276351) served as an outgroup to root the tree. GenBank accession numbers for the submitted 16S rRNA gene sequences are JX503935-JX504008.

Primers viz. PR For and PR Rev were designed on the basis of the submitted nucleotide sequence (KF767352) for amplification of LIR (Table 1). The amplification was performed in 50 μl of reaction mixture containing 100 ng of template DNA (RCA product), 50 pmol of each primer, 1x Taq DNA polymerase buffer, 200 μM dNTPs and 1.25 U AmpliTaq DNA Polymerase (Applied Biosystems, USA). The reactions were conducted at 94°C for 5 min as initial denaturation followed by 30 cycles of 94°C for 1 min, 48°C for 2 min, 72°C for 1 min and final extension at 72°C for 5 min. The amplified DNA product was run on 1.5% agarose gel, stained with EtBr and visualized in Gel documentation system (BIO-RAD, USA). The PCR product of the expected size ca. 350 bp was recovered from the agarose gel and purified by using QIAEXII Gel Extraction Kit (QIAGEN, Germany). The quantity and purity of gel eluted DNA were measured by BioPhotometer plus (Eppendorf, Germany) and cloned into pDrive PCR cloning vector (QIAGEN, Germany). The positive colonies were confirmed by PCR and restriction digestion, and nucleotide sequences were determined using an automated sequencer (ABI3730XL, Applied Biosystems, USA).

Genomic DNAs were extracted using the Qiagen DNeasy Blood and Tissue Kit. The 16S rRNA gene was amplified by PCR using the universal bacterial 16S rDNA primers F27 and R1492 (Lane 1991 ). Obtained PCR products were cloned into the Qiagen pDrive PCR cloning vector and sequenced using standard M13 vector primers at MWG Biotech (Germany). DNA sequences of almost complete 16S rRNA genes (ca 1400 nucleotides) were compared with those of the type strains of the genus Actinoalloteichus available in EzTaxon server database (http://www.ezbiocloud.net/identify) (Chun et al. 2007 (link)). Sequences were aligned using Clustal W algorithm (Thompson et al. 1997 (link)) and a phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) software version 7 (Kumar et al. 2016 (link)). The tree was computed using maximum likelihood method (ML) and the resulting tree topology was tested by bootstrap analysis performed with 5000 replicates. The 16S rRNA gene sequences for strains ADI 127-17^T and GBA 129-24 have been deposited in Genbank with the accession numbers MF440323 and MF440324, respectively.