The 16S rRNA gene of the phosphate solubilizing bacterium was amplified using universal 27F forward primer (5′AGGCCTAACACATGCAAGTC-3′) and 1492R reverse primer (5′GGGCGGWGTGTACAAGGGC- 3′), as described by Das et al. [12] (link). PCR product was purified using QIAquick@ gel extraction kit, QIAGEN, GmbH (Germany) and nucleotide sequence were determined using the BigDye Terminator v 3.1 Cycle sequencing kit in an automated 3130xl Genetic Analyzer System (Applied Biosystems, HITACHI, USA). Forward and reverse sequences were aligned using seqscape software (Applied Biosystems, USA) to get the final sequence (585 bp). The final partial sequence was submitted to NCBI gene bank in order to obtain the corresponding accession number. A phylogenetic tree was constructed using MEGA 4.0 software, using the neighbour- joining DNA distance algorithm with a bootstrap of 1000.
3130xl genetic analyzer system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 3130xl Genetic Analyzer System is a capillary electrophoresis-based instrument designed for DNA sequencing and fragment analysis applications. It features a 16-capillary array and can generate high-quality sequencing data and accurate fragment analysis results.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Seqscape software, by Thermo Fisher Scientific (1 mentions)
Sequencing analysis 5, by Thermo Fisher Scientific (1 mentions)
Big dye terminator kit v3, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Superscript 3 one step rt pcr system with platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Pcr clean up column kit, by Qiagen (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
4 protocols using 3130xl genetic analyzer system
1
Molecular Identification of Phosphate Solubilizing Bacteria
2
Validating Genetic Variants via Sanger Sequencing
3
Nested RT-PCR amplification and sequencing
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We amplified a major fragment of reading frame 2 (nt positions 5145‒7127) by using a nested reverse transcription nested RT-PCR. We performed reverse transcription and the first PCR by using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, https://www.thermofisher.com ) and 0.35 nmol/L of each primer (forward: 5′-CCGACAGAATTGATTTCGTCGGC-3′ and reverse: 5′-ACTCCCGRGTYTTACCYACCTT-3′). We performed a nested PCR by using Platinum Taq DNA Polymerase (Invitrogen), 0.32 nmol/L of each primer (forward: 5′-TCGTCTCAGCCAATGGCGAGCC-3′ and reverse: 5′-CASARAANGTCTTNGARTACTGCT-3′), and 30 cycles of standard PCR conditions. We loaded amplicons onto a 2% agarose gel, subjected the amplicons to electrophoresis, and purified specific bands by using the QIAquick Gel Extraction Kit (QIAGEN, https://www.qiagen.com ). We sequenced purified material by using the Sanger method and a 3130xL Genetic Analyzer System (Applied Biosystems, https://www.thermofisher.com ).
4
Bacterial 16S rRNA Sequencing and Phylogenetic Analysis
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Bacterial genomic DNA was isolated from bacterial culture of selected bacterial isolate using Wizard Genomic DNA Purification Kit (Promega, USA) according to the manufacture instructions. By using 16 rRNA specific primers, forward (5`-AGAGTTTGATCCTGGCTCAG-3`) and reverse (5`-GGTTACCTTGTTACGACTT-3`), PCR reaction was performed as previously reported96 (link). Briefly, PCR reaction was started with initial denaturation at 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 50 °C for 30 s and 72 °C for 1.5 min. An additional final extension step was carried out at 72 °C for 5 min. PCR amplified products were checked on 1.5% agarose gel electrophoresis, visualized under UV transilluminator, and purified by a PCR clean-up column kit (QIAGEN, Germany) for sequencing. Sanger sequencing of 16 rRNA gene was performed using a BigDye Terminator v3.1 Cycle Sequencing kit and a 3130xl Genetic Analyzer system (Applied Biosystems, USA). After the sequencing process, the annotated nucleotide sequence was analyzed using NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and deposited in Genbank. The phylogenetic tree was constructed based on the UPGMA statistical method with a bootstrap of 2.000 replicates using the MEGA 5 software97 (link).
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