The bacterial genomic DNA was extracted with the help of HiPurA™ kit (Himedia, India). The bacterial 16S rRNA genes were amplified by PCR using two universal primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′), and 1492R (5′-CGGTTAC CTTGTTACGACTT-3′) in 50µL PCR tube using 4µL each dNTP, 2µL MgCl2, 2µL template DNA, 1µL each primer (forward and reverse), 1µL Taq DNA polymerase, and 33µL nuclease-free water (Himedia, India). Reactions were performed in the MasterCycler gradient (Eppendorf, India) with the following reaction conditions; 94 °C (5 min) for early denaturation steps followed by 30 cycles of 94 °C (30 s), 55 °C (1 min), 72 °C (1 min), and extension at 72 °C (10 min). The purification of the PCR products were done by using HiPurA™ PCR clean up system kit (Himedia, India) and the sequencing was done through Applied Biosystems ABI (3500 Genetic Analyzer, Japan) using each universal primer (27F and 1492R) (Sherpa et al., 2018 (link)). The sequences were assembled and aligned with the aid of the Codon-Code Aligner software. The sequences were identified using NCBI BLASTn and the phylogenetic tree was created by using the neighbor-joining method using MEGA v.10 (Erickson, 2010 (link); Saitou and Nei, 1987 (link)). Gene bank accession was obtained post Bankit submission.
Hipura kit
Manufactured by HiMedia
Sourced in India
The HiPurA™ kit is a nucleic acid purification system designed for the isolation and purification of DNA and RNA from various biological samples. The core function of the kit is to efficiently extract and purify nucleic acids, ensuring high-quality samples for downstream applications such as PCR, sequencing, and molecular biology experiments.
Automatically generated - may contain errors
5 protocols using hipura kit
1
Bacterial 16S rRNA Gene Sequencing Protocol
2
Molecular Characterization of Carbapenemase-Producing Isolates
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The interpretation of combined disc test (Klebsiella pneumoniae carbapenemase + metallo-betalactamases) kit Finally, the molecular characterization was done with polymerase chain reaction (PCR) by the setting of simultaneous detection of common genes responsible for carbapenemases production in all IPM-resistant isolates. The presence of MBL-producing genes VIM, NDM, and IMP was evaluated. Furthermore, KPC was assessed, as together, these are the most frequently encountered carbapenemases.[ 16 ] Control strains were used as positive and negative controls. DNA was extracted (HiPurA™ kit, HiMedia) and amplified in a thermocycler (Touchgene Gradient, TECHNE). Multiplex and uniplex PCR were performed on all carbapenem-resistant isolates. The separated DNA fragments on agarose gel electrophoresis with specific bands at particular gene amplicon size were considered as positive PCR reactions.[ 1718 ] The primers (Eurofins Genomics) used and DNA fragments of a specific size are depicted in Table 2.
3
Fungal Mycelium Genomic DNA Extraction
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Fungal mycelium from each pure culture was aseptically transferred to the broth culture medium and incubated in a BOD incubator at 28 ± 2°C for one week. The genomic DNA was extracted from approximately 100 mg of fresh mycelium by crushing it in micro centrifuge tubes using micro-pestles in liquid nitrogen. The HiPura kit of HiMedia Company and protocols suggested by Birren & Lai 1993; Sambrook et al., 1989 were followed for genomic DNA isolation. The DNA was eluted in 200 µl of Tris EDTA buffer (TE buffer). The yield and purity of extracted DNA was determined electrophoretically on 0.8 % agarose gel and spectrophotometrically at 260 and 280 nm wavelength using BioPhotometer (D30, Eppendorf Germany)
4
Molecular Characterization of Fungal and Bacterial Isolates
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The PCR amplified full length DNA from both, the fungus (~540 bp) and the bacteria (~1500 bp) were confirmed in a 1.5% agarose gel and purified using the Hipur A kit (Himedia, India) as specified by the manu-facturerʼs protocol and custom sequenced using the facility at Eurofins Genomics Private Limited, Bengaluru, India. The obtained gene sequences were then submitted to a BLAST search to obtain the best matching sequences. For phylogenetic analyses, reference sequences required for comparison were downloaded from the Genbank database (http://www.ncbi.nlm.nih. gov/Genbank) . Sequences were aligned with the aid of the multiple sequence alignment program CLUSTAL W. The aligned sequences were then checked for gaps manually, and saved in the molecular evolutionary genetics analysis (MEGA) format using the MEGA 5 software. To obtain the confidence values, the original data set was resampled 1000 times using the bootstrap analysis method and used directly for constructing the phylogenetic tree. The phylogenetic trees were constructed using the neighbor-joining method. All these analyses were performed with the aid of Mega 5 software (Tamura et al., 2011) .
5
Fungal Genomic DNA Isolation
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For DNA isolation, a small piece of growing mycelia from each of the 52 isolated cultures was aseptically transferred to separate Malt Extract Dextrose Broth culture medium and incubated in a BOD incubator at 25 ± 2 °C for seven days. The fungal mycelial net from each raised pure culture was filtered using filter paper through funnel. The genomic DNA was extracted from approximately 100 mg of fresh mycelium by crushing it in conical micro centrifuge tubes using micropestles in liquid nitrogen. The HiPura kit of HiMedia Company and protocols suggested by Birren and Lai (1993) , Sambrook, Fritsch and Maniatis (1989) were followed for genomic DNA isolation. Finally, the DNA was eluted in 200µL of Tris EDTA buffer (TE buffer).
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